bromochloroacetic-acid and involucrin

To investigate the molecular mechanism of pathological keratinization in severe ocular surface diseases based on the process of epidermal physiological keratinization and the gene expression profile of ocular surface epithelium.. We examined conjunctiva covering corneas which had ocular surface diseases in which pathological keratinization was seen. We then investigated the expression of epidermal keratinization-related proteins and clusterin, which is the most abundant gene transcript in human corneal epithelium.. Semiquantitative reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry showed that transglutaminase 1, involucrin, filaggrin, and cytokeratin 1/10 expression was upregulated in keratinized conjunctiva compared to normal conjunctiva. On the other hand, the level of clusterin expression in the diseased ocular surfaces was significantly lower than normal. We also concluded that clusterin expression may depend on the severity of the pathological keratinization.. Various keratinization-related proteins and clusterin are most likely to be involved in the pathogenesis of cicatrizing ocular surface diseases.

Topics: Clusterin; Conjunctiva; Conjunctival Diseases; Filaggrin Proteins; Glycoproteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Molecular Chaperones; Protein Precursors; Transglutaminases

The epidermis is a tissue that undergoes a very complex and tightly controlled differentiation program. The elaboration of this program is generally flawless, resulting in the production of an effective protective barrier for the organism. Many of the genes expressed during keratinocyte differentiation are expressed in a coordinate manner; this suggests that common regulatory models may emerge. The simplest model envisions a 'common regulatory element' that is possessed by all genes that are regulated together (e.g., involucrin and transglutaminase type 1). Studies to date, however, have not identified any such elements and, if anything, the available studies suggest that appropriate expression of each gene is achieved using sometime subtly and sometime grossly different mechanisms. Recent studies indicate that a variety of transcription factors (AP1, AP2, POU domain. Sp1, STAT factors) are expressed in the epidermis and, in many cases, multiple members of several families are present (e.g., AP1 and POU domain factors). The simultaneous expression of multiple members of a single transcription factor family provides numerous opportunities for complex regulation. Some studies suggest that specific members of these families interact with specific keratinocyte genes. The best studied of these families in epidermis is the AP1 family of factors. All of the known AP1 factors are expressed in epidermis [52] and each is expressed in a specific spatial pattern that suggests the potential to regulate multiple genes. It will be important to determine the role of each of these members in regulating keratinocyte gene expression. Finally, information is beginning to emerge regarding signal transduction in keratinocytes. Some of the early events in signal transduction have been identified (e.g., PLC and PKC activation, etc.) and some of the molecular targets of these pathways (e.g., AP1 transcription factors) are beginning to be identified. Eventually we can expect to elucidation of all of the steps between the interaction of the stimulating agent with its receptor and the activation of target gene expression.

Topics: Animals; Cell Division; DNA-Binding Proteins; Epidermis; Gene Expression Regulation, Developmental; Homeodomain Proteins; Humans; Keratinocytes; Keratins; Membrane Proteins; Protein Kinase C; Protein Precursors; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcription Factor AP-1; Transcription Factor AP-2; Transcription Factors

Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Disease Progression; Epidermal Cells; Epithelial Cells; Humans; Keratins; Neoplasm Proteins; Neoplasms; Protein Precursors; Skin Neoplasms; Tumor Cells, Cultured

This review summarizes recent data on the keratinocyte, the major cell type in the epidermis. A two-state model is proposed: in the "resting" state, the keratinocyte is committed to a program of terminal differentiation focusing on production of an efficient barrier, the stratum corneum, and of the epidermal basement membrane; the activated state, first discovered in cell culture studies, occurs in vivo during epidermal wound healing and inflammatory skin diseases.

Topics: Cell Differentiation; Epidermal Cells; Epidermis; Filaggrin Proteins; Humans; Interleukin-1; Intermediate Filament Proteins; Keratinocytes; Keratins; Protein Precursors; Skin Diseases; Transglutaminases; Wound Healing

Topics: Antibodies, Monoclonal; Antigens, Differentiation; Biomarkers, Tumor; Carbonic Anhydrases; Carcinoembryonic Antigen; CD57 Antigens; HLA Antigens; Humans; Immunohistochemistry; Keratins; Membrane Glycoproteins; Mucin-1; Protein Precursors; S100 Proteins; Sweat Gland Neoplasms

Epidermal keratinocytes (skin cells) are highly specialized epithelial cells designed to perform a very specific function, separation of the organism from its environment. To accomplish this the cells synthesize precursors and assemble them into two distinct structures, the cornified envelope and keratin intermediate filaments. The intermediate filaments are assembled from keratin monomers and the cornified envelope is assembled from a protein called involucrin and several other proteins. Expression of involucrin and the keratins genes are regulated as a function of the stage of keratinocyte differentiation and by various external agents such as calcium and vitamin A. To study the function of these structures and the regulation of precursor production we have cloned cDNA and genomic clones encoding involucrin and four of the keratin polypeptides. Retinoids profoundly alter the differentiation pattern of human epidermal keratinocytes, but the underlying biochemical basis of this change is not known. In this report we describe retinoid-promoted changes in keratin gene expression that may, in part, be responsible for the alteration in cellular phenotype in the presence of the vitamin. We also describe the novel structure of the human 40 kD keratin, a member of the keratin family that is retinoid responsive and is likely to be important during epidermal development. Finally, we describe the structure of the envelope precursor protein, involucrin, as determined from its DNA sequence and speculate on its role in cornified envelope formation.

Topics: Animals; Cell Differentiation; Epidermal Cells; Gene Expression Regulation; Humans; Keratins; Protein Precursors; Retinoids

The mechanisms of action of urea-containing ointments in the treatment of eczema, ichthyosis and psoriasis are only partly known and related to proteolysis and keratinolysis. In this study, we have examined the effects of topical urea on epidermal proliferation and differentiation in 10 patients with psoriasis. Plaque type lesions were treated for 2 weeks with an ointment containing 10% urea, with the vehicle alone, or left untreated. Clinical score, hydration of the stratum corneum, transepidermal water loss (TEWL), and immunohistochemical studies were performed. Epidermal proliferation was assessed using the Ki-S3 proliferation-associated nuclear antigen. For epidermal differentiation antibodies against involucrin and against keratins CK 5, 6, 17 and CK 1, 5, 10, 14 were used. The patients showed a reduction of the clinical score (> 50%), a 2-fold increase in stratum corneum hydration (p < 0.01), and a small decrease in TEWL (N.S.) on the urea- treated compared to the untreated site. Light microscopy studies revealed a 29% reduction in epidermal thickness (p < 0.01); epidermal proliferation was decreased by 51% (p < 0.005). The altered expression of involucrin and of cytokeratins (reduction of CK 5, 1 and 10 and induction of CK 6 and 17) was partially reversed. The ointment base also improved psoriasis, but urea was significantly better than the vehicle (urea: 40% reduction in epidermal proliferation vs. vehicle). In summary, these studies show that urea influences epidermal proliferation and differentiation in psoriasis.

Topics: Administration, Topical; Adolescent; Cell Differentiation; Cell Division; Double-Blind Method; Epidermis; Humans; Immunohistochemistry; Keratins; Ointments; Protein Precursors; Psoriasis; Urea

Oral retinoids have been widely used in psoriasis, but topical forms have been ineffective or irritating.. Our purpose was to determine the clinical and molecular effects of a new topical retinoid, AGN 190168, on psoriasis.. Seven patients with psoriasis were treated for 2 weeks with topical retinoid and 2 weeks with vehicle. Two control subjects with psoriasis were treated for 2 weeks with vehicle alone. Biopsy specimens from normal skin as well as from untreated and treated psoriatic lesions were compared by immunohistochemical analysis. Differentiation and inflammatory markers were studied.. Clinical improvement was seen in all seven patients after 2 weeks of treatment. Improvement was still present, but not significant, after 2 additional weeks of vehicle application. Histologic examination showed a return to a more normal morphology in four of seven biopsy specimens, which correlated with filaggrin expression. There was a diminution in the precocious expression of keratinocyte transglutaminase, keratin 16, and involucrin, as well as a decrease in epidermal growth factor receptor and in the number of cells expressing intercellular adhesion molecule type 1 and HLA-DR.. Clinical and histologic improvements were seen in psoriasis in association with the topical application of AGN 190168 at 2 weeks, including decreased inflammation and restoration of normal epidermal differentiation. Small patient numbers and the possibility that the changes were related to clinical improvement alone and not the topical agent preclude definitive conclusions.

Topics: Administration, Cutaneous; Adult; Antigens, CD; Biopsy; Cell Adhesion Molecules; Double-Blind Method; Epidermis; ErbB Receptors; Female; Filaggrin Proteins; Follow-Up Studies; HLA-DR Antigens; Humans; Immunohistochemistry; Intercellular Adhesion Molecule-1; Intermediate Filament Proteins; Keratinocytes; Keratins; Male; Nicotinic Acids; Pilot Projects; Prospective Studies; Protein Precursors; Psoriasis; Retinoids; Severity of Illness Index; Skin; Transglutaminases

Histologic and immunocytochemical analyses were performed on cutaneous biopsies from 10 patients treated with retinoic acid under occlusion for 4 d compared to biopsies from 19 patients treated nightly for 16 weeks. Acute application of RA caused epidermal thickening (9 of 10 samples), stratum granulosum thickening (7 of 10), parakeratosis (4 of 10), a marked increase in the number of cell layers expressing epidermal transglutaminase (7 of 10), and focal expression of two non-epidermal keratins, K6 (8 of 10) and K13 (2 of 10), changes also observed with chronic treatment. Involucrin, filaggrin, and loricrin were also altered in samples from both acute and chronic treatment. An increased number of cell layers expressed both involucrin and filaggrin from both the acute (7 of 10) and chronic (14 of 19) treatment groups. In the acute group, loricrin expression was significantly reduced or absent in some regions of the epidermis (5 of 10), whereas most chronic samples showed an increased number of cell layers expressing loricrin (12 of 19). The pattern of expression of three major epidermal differentiation products, keratins K1, K10, and K14, was not significantly altered in any of the acute or chronic samples, although there was a slight reduction in the detection of K10 in two of the acute samples. Thus, acute topical RA treatment under occlusion caused substantial changes in the epidermis, and reproduced most, but not all of the effects of chronic treatment.

Topics: Administration, Topical; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Membrane Proteins; Protein Precursors; RNA, Messenger; Skin; Transglutaminases; Tretinoin

Benzalkonium chloride is the most widely used preservative in ophthalmic topical solutions. The aim of this study was to investigate the influence of BAC as a single substance or as a component of several commercially available ophthalmic solutions on meibomian gland epithelial cells in vitro.. An immortalized human meibomian gland epithelial cell line (HMGEC) was used and cells were cultured in the absence or presence of fetal bovine serum to assess cell morphology, cell proliferation, cell viability (MTS assay) and impedance sensing (ECIS) after stimulation with BAC. Further, the viability of HMGECs stimulated with BAC-containing and BAC-free bimatoprost, travoprost and latanoprost was evaluated using the MTS assay. Real-time PCR analysis for hyperkeratinization associated genes (cornulin, involucrin) was performed.. In the absence of serum, the proliferation rate of HMGECs decreased starting with 0.1μg/ml BAC. At concentrations of 50μg/ml BAC and higher, cell viability was reduced after 10min exposure with a corresponding change in cell morphology. Toxicity of BAC-containing ophthalmic solutions was greater than that of BAC alone, whereas BAC-free alternative products did not significantly influence cell viability. Confluence, cell-cell contacts and serum-containing medium appeared to facilitate HMGECs survival. Expression rate of involucrin and cornulin declined after exposure to preserved bimatoprost and BAC.. BAC showed cytotoxic effects on HMGECs starting with a concentration of 0.1μg/ml. The combination of BAC and prostaglandin-analogs might have a synergistic effect which results in higher toxicity than BAC alone. Unpreserved eye drops and eye drops preserved with Polyquaternium-1 are less damaging to HMGECs.

Topics: Benzalkonium Compounds; Cell Line; Cell Proliferation; Cell Survival; Drug Synergism; Epithelial Cells; Humans; Keratins; Meibomian Glands; Ophthalmic Solutions; Preservatives, Pharmaceutical; Prostaglandins; Protein Precursors; Real-Time Polymerase Chain Reaction

Percutaneous osseointegrated prosthetics (POP), which consist of a metallic post attached to the bone that extends outward through the skin to connect to an external prosthesis, have become a clinically relevant option to replace the typical socket-residual limb connection. POP devices offer several advantages such as mechanical off-loading of soft tissues, direct force transfer to the musculoskeletal system, greater proprioception, and overall improvement in limb kinesis compared to a socket system. However, POP devices create several challenges including epidermal downgrowth, increased infection risk, and mechanical tearing at the skin-implant interface. To address these issues, biomimetic surfaces and coatings have been developed in an attempt to create an infection-free and cohesive interface between POP devices and skin. The fingernail is a prime example of a natural system with a skin interface that is both mechanically and biologically stable. Exploiting keratins' previously demonstrated tissue compatibility and creating a biomimetic coating for POP devices that can imitate the human fingernail, and demonstrating its ability to promote a stable interface with skin tissue is the goal of this work. Silane coupling aided in producing a coating on titanium substrates consisting of human keratin proteins. Several combinations of silane and keratin derivatives were investigated, and in general showed a nano-scale coating thickness that supported skin cell (i.e. fibroblast and keratinocyte) adhesion. Initial enzyme-mediated degradation resistance was also demonstrated, but the coatings appeared to degrade at long time periods. Importantly, keratinocytes showed a stable phenotype with no indication of wound healing-like activity. These data provide justification to further explore keratin biomaterials for POP coatings that may stabilize the implant-skin interface.

Topics: Actins; Biomarkers; Biomimetic Materials; Cell Adhesion; Cell Line; Coated Materials, Biocompatible; Fibroblasts; Gene Expression; Hair; Humans; Keratinocytes; Keratins; Prostheses and Implants; Protein Precursors; Silanes; Titanium

Oral lichen planus (OLP) characterized by interface mucositis frequently shows hyper-keratinization. To clarify mechanisms of excess keratinization, we investigated key molecules for cornified cell envelope (CE).. Involucrin (IVL), loricrin (LOR), transglutaminase 1 (TGase 1) and transglutaminase 3 (TGase 3) were immunohistochemically examined in 20 specimens of OLP; five specimens of buccal mucosa served as controls. Subsequently, the data were statistically analyzed.. IVL in OLP was localized in the cell membrane, in contrast to its localization in the cytoplasm in controls. No positive reaction indicative of LOR was noted in any specimens. Although the TGase 1 localization in controls was restricted to the upper three-quarters of the membrane, the localization in OLP was in both membrane and in the cytoplasm of full thickness mucosal layers. The TGase 3 localization pattern was dramatically altered from cytoplasmic to membranous in OLP.. Our data suggest that aberrant TGase 1 and TGase 3 localization and distribution are closely related to hyper-keratinization in OLP. This is the first report of ectopic transglutaminase localization in OLP.

Topics: Cell Membrane; Epithelium; Humans; Intracellular Space; Keratins; Lichen Planus, Oral; Middle Aged; Protein Precursors; Transglutaminases

Symmetrical acrokeratoderma (SAK) is characterized by brown to black hyperkeratotic patches on acral regions. Although epidermal hyperkeratosis and acanthosis are consistent pathological changes, the nature of epidermal hyperplasia is unknown.. To evaluate epidermal proliferation and differentiation and melanocytic density in skin lesions of SAK.. Expression of keratin 10 (K10), K14, K16, involucrin, filaggrin, Ki-67, and Melan-A was detected by immunohistochemistry in eight patients with SAK, seven patients with ichthyosis vulgaris (IV) and six healthy controls (HCs).. Expression of K14, K16, involucrin and filaggrin was upregulated in patients with SAK compared with patients with IV and the HCs (P < 0.01-0.05), but K10 expression was similar for the three groups (P > 0.05). Numbers of Ki-67+ and Melan-A+ cells were higher in patients with SAK than in patients with IV and the HCs (P < 0.05).. These results demonstrate that excessive keratinocyte proliferation and abnormal differentiation contribute to epidermal hyperplasia, while melanocytic proliferation is responsible for the pigmented lesions in SAK.

Topics: Adolescent; Adult; Cell Differentiation; Cell Proliferation; Epidermal Cells; Epidermis; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratosis; Ki-67 Antigen; Male; Melanocytes; Protein Precursors; Skin; Up-Regulation; Young Adult

Here, we report that siRNA transfection of β-adducin significantly disrupted the spectrin-based cytoskeleton and cytoskeletal arrangements of both β-adducin and PKCδ by substantially inhibiting the expression of β-adducin, spectrin and PKCδ proteins in differentiating keratinocytes. However, extracellular Ca2+ treatment blocked the inhibitory effects of the β-adducin siRNA. Ca2+ also prevented the significant down-regulation of two differentiation markers involucrin and K1/10 and the distinct up-regulation of proliferation marker K14 in β-adducin siRNA transfected keratinocytes. In addition, β-adducin knockdown resulted in a substantial reduction of epidermal growth factor receptor (EGFR), cadherin and β-catenin and enhanced phosphorylation of EGFR on tyrosine 1173 and Ca2+ prevented these changes. Furthermore, Ca2+ blocked the inhibitory effects of β-adducin siRNA on the expression of calmodulin, phosphorylated-calmodulin (P-CaM((Tyr138))) and myristoylated alanine-rich C-kinase substrate (MARCKS) in keratinocytes. Co-immunoprecipitation studies further revealed that calmodulin, not MARCKS, strongly interacted with EGFR, cadherin and β-catenin. Our data suggest that Ca2+ plays an important role in regulating the expression and function of β-adducin to sustain normal organization of the spectrin-based cytoskeleton and the differentiation properties in keratinocytes through the calmodulin/EGFR/cadherin signaling pathway.

Topics: Animals; Cadherins; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cell Differentiation; Cell Shape; Cytoskeleton; ErbB Receptors; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Keratinocytes; Keratins; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Precursors; Proto-Oncogene Proteins c-akt; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spectrin; src-Family Kinases; Transfection

Dental alloys containing indium (In) have been used in dental restoration for two decades; however, no study has investigated the biological effects of In ions, which may be released in the oral cavity, on human oral keratinocytes. The objective of the present study was to investigate the biological effects of In ions on human oral keratinocyte after confirming their release from a silver-palladium-gold-indium (Ag-Pd-Au-In) dental alloy.. As a corrosion assay, a static immersion tests were performed by detecting the released ions in the corrosion solution from the Ag-Pd-Au-In dental alloy using inductively coupled plasma atomic emission spectroscopy. The cytotoxicity and biological effects of In ions were then studied with In compounds in three human oral keratinocyte cell lines: immortalized human oral keratinocyte (IHOK), HSC-2, and SCC-15.. Higher concentrations of In and Cu ions were detected in Ag-Pd-Au-In (P<0.05) than in Ag-Pd-Au, and AgCl deposition occurred on the surface of Ag-Pd-Au-In after a 7-day corrosion test due to its low corrosion resistance. At high concentrations, In ions induced cytotoxicity; however, at low concentrations (∼0.8In(3+)mM), terminal differentiation was observed in human oral keratinocytes. Intracellular ROS was revealed to be a key component of In-induced terminal differentiation.. In ions were released from dental alloys containing In, and high concentrations of In ions resulted in cytotoxicity, whereas low concentrations induced the terminal differentiation of human oral keratinocytes via increased intracellular ROS. Therefore, dental alloys containing In must be biologically evaluated for their safe use.

Topics: Blotting, Western; Cell Differentiation; Corrosion; Dental Alloys; Electrochemical Techniques; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Fibronectins; Gold Alloys; Humans; Indium; Ions; Keratinocytes; Keratins; Materials Testing; Palladium; Protein Precursors; Reactive Oxygen Species; Silver; Spectrophotometry, Atomic; X-Ray Diffraction

Neuronatin (Nnat), which is a neuronal developmental and differentiation molecule, is expressed in the endoplasmic reticulum of non-neuronal cells and is involved in insulin secretion from pancreatic β-cells by plausibly modulating their intracellular calcium concentration. However, the role of Nnat in keratinocyte differentiation remains unclear.. To unveil a possible integration of Nnat in controlling the keratinocyte differentiation markers such as involucrin, cytokeratin1, filaggrin, loricrin and S100A7.. Immunohistological staining was done using psoriasis, chronic eczema, lichen planus and normal skin. Immunofluorescence staining, Western blotting and semi-quantitative real-time PCR were performed for detecting Nnat, involucrin, cytokeratin1, filaggrin, loricrin and S100A7 using human keratinocytes with or without Nnat gene transfection. Small interference RNA was applied to knockdown the Nnat gene expression.. Nnat existed in normal human epidermis and cultured keratinocytes. In the hyperplastic epidermis of psoriasis, chronic eczema and lichen planus, over-expression of Nnat was evident along with involucrin and cytokeratin1 expression. Coordinate up-regulation of Nnat and involucrin, but not cytokeratin1, was demonstrated in cultured keratinocytes under differentiation stimuli such as extracellular calcium elevation, exposure to phorbol myristate acetate, and increased cell density. Transfection of small intereference RNA for Nnat decreased the mRNA levels of Nnat and involucrin, but not of cytokeratin1. Furthermore, a gene transfection assay showed increased involucrin expression in the Nnat-transfected keratinocytes than in mock-transfected counterparts, without any appreciable influence on cytokeratin1, filaggrin, loricrin and S100A7 expression.. These data indicate that Nnat is related to keratinocyte differentiation by up-regulating involucrin expression.

Topics: Cell Differentiation; Cells, Cultured; Filaggrin Proteins; Humans; Keratinocytes; Keratins; Membrane Proteins; Nerve Tissue Proteins; Protein Precursors; Up-Regulation

Barrett's esophagus is characterized by a distinct Th2-predominant cytokine profile (IL-4) from in vivo or ex vivo evidence. The detailed role of cytokines in Barrett's esophagus, particularly whether Th2 cytokines are causative factors driving metaplastic processes, remains unknown. In this study, air-liquid interface-cultured human esophageal epithelial cells were stimulated by a Th2 cytokine, IL-4, and Th1 cytokines, TNF-α and IL-1β, continuously for 10 days. Barrier function was determined by transepithelial electrical resistance. Morphological changes were investigated by hematoxylin and eosin staining. Keratin profile (keratin 7, 8, 13, and 14) and squamous differentiation markers (involucrin) were investigated by RT-quantitative PCR, Western blotting, and immunohistochemical staining. Pharmacological inhibitors were used to identify the underlying cellular signaling. We report that IL-4, TNF-α, and IL-1β decrease barrier function, but only IL-4 significantly increases cell layers and changes cell morphology. IL-4 time dependently downregulates the expression levels of the squamous cell markers involucrin and keratin 13 and upregulates the expression levels of the columnar cell markers keratin 7 and 8. Neither TNF-α nor IL-1β shows any effect on these indexes. JAK inhibitor I and PI3K inhibitors significantly block the IL-4-induced changes in the levels of keratin 8 and 13. In conclusion, IL-4 inhibits squamous differentiation program of esophageal epithelial cells and induces differentiation toward columnar cells through the JAK/PI3K pathway. Thus IL-4 may be involved in the early stages of Barrett's esophagus development.

Topics: Barrett Esophagus; Biomarkers; Cell Differentiation; Cells, Cultured; Epithelial Cells; Esophagus; Humans; Immunohistochemistry; Interleukin-1beta; Interleukin-4; Keratins; Metaplasia; Protein Precursors; Signal Transduction; Tumor Necrosis Factor-alpha

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo.. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model).. Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro.. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro.

Topics: Animals; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Culture Media; Dermatologic Surgical Procedures; Epithelial Cells; Fibroblasts; Fluorescent Dyes; Keratin-18; Keratin-19; Keratin-8; Keratins; Laminin; Male; Mice; Mice, Nude; Organic Chemicals; Periodontal Ligament; Protein Precursors; Swine; Time Factors; Tissue Culture Techniques; Tissue Engineering; Tissue Scaffolds

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.

Topics: Acyclovir; Antiviral Agents; Cell Culture Techniques; Cell Differentiation; Cytopathogenic Effect, Viral; DNA, Viral; Epstein-Barr Virus Nuclear Antigens; Gene Expression Regulation, Viral; Gingiva; Herpesvirus 4, Human; Humans; Keratinocytes; Keratins; Palatine Tonsil; Plasmids; Protein Precursors; RNA, Viral; Trans-Activators; Viral Matrix Proteins; Viral Proteins; Virus Cultivation; Virus Latency; Virus Replication

Perinatal stem cells such as human umbilical cord Wharton's jelly stem cells (HWJSCs) are excellent candidates for tissue engineering because of their proliferation and differentiation capabilities. However, their differentiation potential into epithelial cells at in vitro and in vivo levels has not yet been reported. In this work we have studied the capability of HWJSCs to differentiate in vitro and in vivo to oral mucosa and skin epithelial cells using a bioactive three-dimensional model that mimics the native epithelial-mesenchymal interaction. To achieve this, primary cell cultures of HWJSCs, oral mucosa, and skin fibroblasts were obtained in order to generate a three-dimensional heterotypical model of artificial oral mucosa and skin based on fibrin-agarose biomaterials. Our results showed that the cells were unable to fully differentiate to epithelial cells in vitro. Nevertheless, in vivo grafting of the bioactive three-dimensional models demonstrated that HWJSCs were able to stratify and to express typical markers of epithelial differentiation, such as cytokeratins 1, 4, 8, and 13, plakoglobin, filaggrin, and involucrin, showing specific surface patterns. Electron microscopy analysis confirmed the presence of epithelial cell-like layers and well-formed cell-cell junctions. These results suggest that HWJSCs have the potential to differentiate to oral mucosa and skin epithelial cells in vivo and could be an appropriate novel cell source for the development of human oral mucosa and skin in tissue engineering protocols.

Topics: Animals; Cell Differentiation; Epithelial Cells; Epithelium; Filaggrin Proteins; Flow Cytometry; Fluorescent Antibody Technique; gamma Catenin; Humans; Intermediate Filament Proteins; Keratins; Leukocyte Common Antigens; Mesenchymal Stem Cells; Mice; Mice, Nude; Models, Biological; Mouth Mucosa; Protein Precursors; Regeneration; Skin; Thy-1 Antigens; Wharton Jelly

Interest in colorless intermediates of melanocyte metabolism has traditionally been related to their role as melanin precursors, though several lines of evidence scattered in the literature suggested that these compounds may exert an antioxidant and protective function per se unrelated to pigment synthesis. Herein, we disclose the remarkable protective and differentiating effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible dopachrome tautomerase (DCT)-dependent eumelanin intermediate, on primary cultures of human keratinocytes. At micromolar concentrations, DHICA induced: (a) time- and dose-dependent reduction of cell proliferation without concomitant toxicity; (b) enhanced expression of early (spinous keratins K1 and K10 and envelope protein involucrin) and late (loricrin and filaggrin) differentiation markers; (c) increased activities and expression of antioxidant enzymes; and (d) decreased cell damage and apoptosis following UVA exposure. The hitherto unrecognized role of DHICA as an antiproliferative, protective, and antiapoptotic endogenous cell messenger points to a reappraisal of the biological functions of melanocytes and DCT in skin homeostasis and photoprotection beyond the mere provision of melanin pigments, and provides, to our knowledge, a previously unreported possible explanation to the higher resistance of the dark-skinned eumelanic phenotypes to sunburn and skin cancer.

Topics: Apoptosis; Cell Communication; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Cells; Epidermis; Filaggrin Proteins; Humans; Indoles; Intermediate Filament Proteins; Keratinocytes; Keratins; Melanins; Melanocytes; Membrane Proteins; Peroxisome Proliferator-Activated Receptors; Protein Precursors; Time Factors

The objectives of this study were to determine (i) the expression of oral cytokeratins (CKs) among human immunodeficiency virus (HIV)-infected subjects compared with non-HIV controls, (ii) the oral CK expression in the subjects with highly active antiretroviral therapy (HAART) compared with those without HAART, and (iii) factors associated with the expression of oral CKs.. Oral tissues from buccal mucosa were obtained by punched biopsy in HIV-infected subjects with and without HAART, and non-HIV individuals. The samples were processed for immunohistochemical studies of CK1, CK13, CK14, CK16, and involucrin. The staining intensity was scored and recorded. Logistic regression analysis and multi-way ANOVA test were performed.. The expression of CK13, CK14, and CK16 was found to be significantly different between HIV-infected subjects and non-HIV individuals (P < 0.05). The expression of those CKs was also significantly different between those who were and were not on HAART (P < 0.05). No significant difference between the groups was observed regarding CK1 and involucrin..  Oral epithelial cell differentiation as marked by the CK expression is affected by HIV infection and use of HAART. CKs may be the useful biomarkers to identify HIV-infected subjects who are at risk of malignant transformation of the oral mucosa because of HIV infection and HAART.

Topics: 3,3'-Diaminobenzidine; Adult; Alcohol Drinking; Antiretroviral Therapy, Highly Active; Biopsy, Needle; CD4 Lymphocyte Count; Chromogenic Compounds; Cross-Sectional Studies; Epithelial Cells; Female; HIV; HIV Infections; HIV Seropositivity; Humans; Keratin-1; Keratin-13; Keratin-14; Keratin-16; Keratins; Male; Middle Aged; Mouth Mucosa; Protein Precursors; Smoking; Viral Load; Young Adult

Canine atopic dermatitis (CAD) is a common allergic skin disease in dogs, associated with a defective epidermal barrier. In this study we investigated the alterations in skin keratinocyte proliferation and differentiation in CAD by quantitative reverse transcription-polymerase chain reaction. Gene expression of keratin (KRT) markers of proliferative and differentiated keratinocytes, together with that of cornified envelope proteins, involucrin (IVL) and filaggrin (FLG), were evaluated. An upregulation of KRT5 and KRT17 in both lesional and non-lesional AD skin was observed (p<0.05) whereas KRT2e, KRT14, IVL and FLG expression were significantly increased only in lesional AD skin (p<0.05). Additionally, the expression levels of KRT5, KRT14, KRT17 and IVL in CAD were strongly correlated. In conclusion, the expression of the majority of the studied keratins, as well as IVL and FLG is increased in CAD with close correlation between the proliferative keratins. This is the first report of a correlation of KRT and IVL genes with CAD.

Topics: Animals; Dermatitis, Atopic; Dog Diseases; Dogs; Filaggrin Proteins; Gene Expression Regulation; Intermediate Filament Proteins; Keratins; Protein Precursors; Skin; Transcriptome

Skin cancers and extrinsic aging are delayed consequences of cumulative UV radiation insults. Exposure of human keratinocytes to UVB has been previously shown to trigger premature senescence. In order to explore the involvement of the cyclin-dependent kinase inhibitor p16(INK-4a) in UVB-induced premature senescence, we developed an original model of repeated sublethal exposures of human keratinocytes deficient in p16(INK-4a). We did not observe any significant increase of senescence-associated beta-galactosidase activity positive cells following UVB exposure in this cell line in contrast to primary keratinocytes, suggesting a role for p16(INK-4a) in UVB-induced senescence. However, we detected sustained DNA damage, prolonged cell cycle arrest, and induction of markers of epidermal differentiation like involucrin and filaggrin as consequences of the repeated exposures. Keratinocytes exposed to the same dose of UVB in a single exposure died. Furthermore, the abundance of the keratins 6, 16 and 17 was increased in keratinocytes exposed repeatedly to UVB suggesting an alternative differentiation. This model allows the induction of a state of differentiation observed in vivo with differentiation uncoupled from premature senescence.

Topics: Apoptosis; beta-Galactosidase; Cell Cycle; Cell Differentiation; Cells, Cultured; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Dose-Response Relationship, Radiation; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Protein Precursors; Tumor Suppressor Protein p53; Ultraviolet Rays

Discovery of the significant impact of filaggrin (FLG) mutations on the genetic predisposition to atopic dermatitis (AD) focused attention on the 1q21 locus, where not only FLG but also other epidermal genes are located. In the present study, we compared 1q21 gene expression in lesional versus nonlesional AD skin.. A real-time quantitative PCR analysis of 10 1q21 genes, selected on the basis of a previous microarray study, was performed in skin biopsies from 33 individuals with AD. Three alternative pathway keratins were also evaluated.. In chronic AD skin lesions, we observed an increase in RNA encoding involucrin, S100 calcium-binding proteins A2 and A7-A9 and small proline-rich region (SPRR) proteins 1A and 2C, with fold changes ranging from 2.0 for S100A2 to 15.4 for S100A8 (p < 0.001, Bonferroni corrected), in parallel to the overexpression of the alternative pathway keratins 6A, 6B and 16. The loricrin (LOR) expression level was significantly decreased in lesional AD skin (fold change 0.5; p < 0.01). The expression of the majority of 1q21 genes and alternative keratins was closely correlated; however, for SPRR1A (and SPRR2C) in lesional skin, the correlation with other genes was lost.. We hypothesize that the deregulated increase in SPRR1A expression in chronic atopic skin lesions reflects an insufficient rise in SPRR transcripts, unable to compensate for the lack of LOR and thus contributing to the persistence of chronic AD skin lesions. Turning off the stress response in the skin may be regarded as a goal in the treatment of AD skin lesions, and SPRR genes might be targets for such an approach.

Topics: Adult; Chronic Disease; Cornified Envelope Proline-Rich Proteins; Dermatitis, Atopic; Female; Filaggrin Proteins; Gene Expression Regulation; Humans; Immunoglobulin E; Intermediate Filament Proteins; Keratins; Male; Membrane Proteins; Middle Aged; Protein Precursors; S100 Proteins; Skin

In this study we generated human skin equivalents (HSEs) under submerged conditions mimicking the aqueous in utero environment and investigated the morphology and differentiation process of the formed epidermis. Further, the skin barrier, which resides in the stratum corneum (SC), was characterized by its lipid content, hydration level, and natural moisturizing factor level. The submerged HSEs showed comparable tissue morphology and similar expression of several differentiation markers and SC lipid composition compared with HSEs grown at the air-liquid interface and native human skin. The SC of the submerged HSEs, however, contained more free water and less natural moisturizing factors compared with the air-exposed counterparts. These results show that the presented cell culture method can be utilized to generate HSEs under submerged conditions to study epidermal formation under aqueous conditions.

Topics: Air; Amniotic Fluid; Biomarkers; Body Water; Cell Differentiation; Cells, Cultured; Female; Fibroblasts; Humans; Keratinocytes; Keratins; Lipid Metabolism; Microscopy, Electron, Scanning; Models, Biological; Pregnancy; Protein Precursors; Skin; Tissue Engineering

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Topics: Adhesins, Bacterial; Antigens, Bacterial; Bacterial Adhesion; Cells, Cultured; Epithelial Cells; Humans; Keratins; Lactococcus lactis; Membrane Proteins; Microscopy, Atomic Force; Protein Binding; Protein Interaction Mapping; Protein Precursors; Staphylococcus aureus

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro. However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro. All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro. This experiment clarified that P. acnes influences the differentiation of NHEK in vitro. As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.

Topics: Acne Vulgaris; Base Sequence; Cell Differentiation; Cells, Cultured; DNA Primers; Filaggrin Proteins; Gene Expression; Humans; In Vitro Techniques; Interleukins; Intermediate Filament Proteins; Keratinocytes; Keratins; Propionibacterium acnes; Protein Precursors; RNA, Messenger; Transglutaminases

Molluscum contagiosum (MC) is a Molluscipox virus infection of keratinocytes with hyperplasia and intracytoplasmic inclusions - the molluscum bodies (MBs). Few papers address cytokeratins (K) profile in MC, mainly focusing terminal keratinization process.. Forty-one MC lesions were subjected to immunohistochemical technique to verify K1, K10, K14, K16, involucrin, filaggrin, E-cadherin and p63 expression. MC immunolabeling pattern was compared to adjacent normal appearing epidermis (ANAE).. In MC and ANAE, K1/K10 were expressed in suprabasal layers, K14 was expressed in basal and suprabasal layers and K16 was expressed through all spinous layer. Involucrin and filaggrin were observed in granular, spinous and in basal layer of ANAE and MC. E-cadherin was present up to the first layers of MC while ANAE exhibited E-cadherin labeling at basal and spinous layers. Basal and spinous layers keratinocytes nuclei, in both MC and ANAE, express p63.. Infection by Molluscipox virus alters keratinocyte differentiation status. The presence of K14 and p63 in spinous layer, as well as early expression of involucrin and filaggrin, associated to a hyperproliferative state disclosed by K16 expression, may be a result of disruption in keratinocytes maturation process. The changes observed at ANAE may represent early events in keratinization disturbance.

Topics: Adolescent; Adult; Biomarkers; Cadherins; Cell Adhesion; Cell Differentiation; Child; Child, Preschool; Filaggrin Proteins; Humans; Immunohistochemistry; Infant; Intermediate Filament Proteins; Keratinocytes; Keratins; Membrane Proteins; Middle Aged; Molluscum Contagiosum; Protein Precursors; Young Adult

Epidermodysplasia verruciformis (EV) is a rare genodermatosis with susceptibility to human papillomavirus (HPV) infection, and high risk of skin cancer considered a model of viral oncogenesis.. Fifteen cases of EV plane wart (PW)-type lesions (EV) and 14 cases of PW in healthy individuals were subjected to immunohistochemical technique for cytokeratins (K) 1, 10, 14, 16, 4, involucrin, filaggrin and e-cadherin.. K1/10 showed retarded or negative expression in EV, being substituted by K14. Expression of K14 occurred in the basal and suprabasal layers in both groups, but in EV, its expression was observed up to the more superficial layers. Both groups showed positivity for K16 and K4, involucrin expression in lower levels of the spinous layer and unaltered filaggrin expression. E-cadherin expression was diminished at the koilocytotic foci of both lesions, more superficially in EV.. Infection by HPV may alter the differentiation status of the epidermis, leading to a major expression of K14, delayed or absent expression of K1/10 and earlier involucrin expression, especially in EV. It also stimulates the expression of K16 and K4. Filaggrin expression is not altered, and e-cadherin is diminished in superficial koilocytotic cells' foci in EV.

Topics: Adult; Cadherins; Epidermodysplasia Verruciformis; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Papillomavirus Infections; Protein Precursors; Skin Diseases

Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6-10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes.. Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers.. The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells.. The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections.

Topics: Blotting, Western; Cell Culture Techniques; Cell Line, Transformed; Claudin-1; Electric Impedance; Epithelial Attachment; Filaggrin Proteins; Flow Cytometry; Fluorescent Antibody Technique; Gingiva; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Membrane Proteins; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Protein Precursors; Tight Junctions; Transfection

The expression of epithelial cell markers in the mouse eyelid and lip was investigated in order to understand the nature of the interactions of mucosal and skin epithelium as to how they form the mucocutaneous junction (MCJ).. Cryosections of eyelid and lip tissue from normal mice were examined immunohistochemically with cytokeratins (CKs): CK1, CK4, CK5, CK6, CK10, CK13, CK14, and CK19; filaggrin; involucrin; and connexin 43.. The expression pattern varied across the MCJ, with the absence of CK1, CK10, and filaggrin in the mucosal epithelium; and CK4, CK6, and CK13 in the skin epidermis. CK5 and CK14 were consistently expressed in full-thickness skin, MCJ, and mucosa. CK19 was expressed basally, while involucrin-positive cells were found superficially in skin, MCJ, and mucosa. Connexin 43 was present in the MCJ, skin, and labial mucosa; however, little to no expression was seen in the palpebral conjunctiva.. The MCJ may be a focal point of mucosal epithelial cell differentiation activities. The similarity of staining patterns in the eyelid and lip suggests that the formation of these sites of shared interaction between the internal and external environment employs similar cellular mechanisms.

Topics: Animals; Biomarkers; Conjunctiva; Connexin 43; Epidermis; Epithelial Cells; Epithelium; Eyelids; Filaggrin Proteins; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Lip; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mucous Membrane; Protein Precursors; Skin; Staining and Labeling; Tissue Distribution

To get a better understanding of the abnormal differentiation or maturation of keratinocytes, we studied the expression and distribution of cytokeratin and involucrin in lesions from systemic lupus erythematosus patients. Two groups of 10 specimens each from systemic lupus erythematosus and normal controls were analyzed by two-dimensional gel electrophoresis, mass spectrometric protein identification, Western blotting and immunohistochemistry. Our results showed that keratin 1 (K1)/K10 together with the new synthesis of K6/K16 were down-regulated and that K5/K14, K2e and involucrin were up-regulated. We found that involucrin was strongly stained in lower epidermal cell layers while K1/10 was weakly stained, particularly when compared with staining in normal epidermis. Additionally, we found that the expression of involucrin was increased. These results imply an aberrant early and terminal differentiation stage in the epidermis of systemic lupus erythematosus, which may be associated with inflammatory cytokines released during the wound healing response of lesion.

Topics: Blotting, Western; Electrophoresis, Gel, Two-Dimensional; Humans; Immunohistochemistry; Keratins; Lupus Erythematosus, Systemic; Protein Precursors; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV).. Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g).. At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer.. Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Topics: Antigens, Differentiation; Cadherins; Epithelium; Gene Expression Regulation, Developmental; Humans; Integrins; Keratins; Protein Precursors; Skin

Tinea corporis is a superficial mycotic infection resulting in substantial epidermal changes. We determined skin barrier function, epidermal differentiation, and human-beta-defensin 2 (hBD-2) protein expression in 10 patients with tinea corporis caused by Trichophyton rubrum (T. rubrum). We found disturbed skin barrier function as shown by a significant increase in transepidermal water loss (TEWL) and specific ultrastructural changes including disturbed formation of extracellular lipid bilayers, lamellar body extrusion, and deposit of clotted material at the stratum granulosum/stratum corneum interface. Epidermal proliferation in tinea increased several fold and accordingly, proliferation and inflammation-associated keratins K6, K16, and K17 were expressed. Expression of basal keratins K5 and K14 increased, whereas differentiation-associated K10 was reduced. Reduction of the cornified envelope proteins involucrin, loricrin, and the S100 protein filaggrin was also seen. Reduced filaggrin expression correlated with reduced skin hydration; protein breakdown products of filaggrin have been shown to be important for water binding. Surprisingly, we found pronounced epidermal protein expression of hBD-2, which may be related to disturbed epidermal differentiation and inflammation. hBD-2 showed a weak, although significant, antifungal activity against T. rubrum in the turbidimetric assay and the immunohistological staining was somewhat less pronounced in areas directly underneath fungal hyphae in the stratum corneum. Together, we describe profound changes in skin barrier structure and function, epidermal proliferation, and differentiation including pronounced protein expression of hBD-2 in tinea corporis.

Topics: beta-Defensins; Cell Differentiation; Cell Membrane Permeability; Cell Proliferation; Dehydration; Epidermis; Filaggrin Proteins; Gene Expression Regulation; Humans; Intermediate Filament Proteins; Keratins; Membrane Proteins; Protein Precursors; Skin Physiological Phenomena; Tinea; Trichophyton

To investigate the differentiation profile of the epithelium of the eyelid margin in the primate.. The expression of cytokeratins (CKs) CK1/10, CK4, CK14, CK5/8, and CK19; involucrin; connexin 43; filaggrin; and muscarinic receptor subtypes (m1-m5) on eyelid tissues from adult Macaca fascicularis (n = 3) was studied by immunohistochemistry.. Cytokeratin 1/10, CK4, CK19, filaggrin, and connexin 43 expression varied across the epithelium of the mucocutaneous junction (MCJ). At the MCJ, CK4-positive cells overlapped basal CK14-expressing cells. The meibomian gland duct and the MCJ revealed layers of CK14-positive cells. Cytokeratin 5/8 was expressed in the basal layer of the epidermis and conjunctiva, while involucrin-positive cells were superficial. The m2 was expressed throughout the conjunctiva; m3 was found in the basal cells of the skin, conjunctiva, and alveolar epithelial cells of the meibomian glands; and m4 was expressed in the suprabasal layers of the skin.. The eyelid margin epithelium is formed from 2 different epithelial cell subpopulations with specific but overlapping distributions. The meibomian gland duct or MCJ may be a site of conjunctival progenitor cells.. This study provides a basis for understanding eyelid pathological conditions and eventually for developing methods for cellular reconstruction of the eyelid margin using expanded progenitor cells.

Topics: Animals; Biomarkers; Cell Differentiation; Conjunctiva; Connexin 43; Epithelial Cells; Eyelids; Filaggrin Proteins; Fluorescent Antibody Technique, Indirect; Intermediate Filament Proteins; Keratins; Macaca fascicularis; Microscopy, Fluorescence; Phenotype; Protein Precursors; Receptors, Muscarinic; Stem Cells

Our previous gene expression analysis suggested that conjunctival epithelial cells of Sjögren's syndrome (SS) are inclined to hyper-proliferation and keratinisation status. The goal of this study is to elucidate whether such pathological situations really exist in the conjunctival epithelium of SS. Also, involvement of inflammatory cytokines in this disease was investigated. Conjunctival tissues or cells obtained from 12 SS patients and 13 normal subjects were subjected to indirect immunostaining to analyse expression of transglutaminase 1 (TGase1), involucrin, keratins 1, 4, 10 and 13. The number of proliferative cells was also analysed by immunostaining of Ki67 antigen. Additionally, changes in gene expression of TGase1 and involucrin after stimulation by IL-1 or IFN-gamma were quantified by real-time RT-PCR. TGase1 and involucrin were up-regulated in the conjunctival epithelium of SS patients. Although not statistically significant, Ki67 positive proliferative cells were slightly increased in SS patients. IFN-gamma stimulation significantly up-regulated TGase1 and unexpectedly repressed involucrin gene expression. IL-1 did not render any significant changes in the expression of these genes. These results suggest the existence of pathological keratinisation in the conjunctival epithelium of SS and also support our hypothesis that inflammatory cytokines may be involved in the ocular surface pathological changes in SS.

Topics: Aged; Biomarkers; Case-Control Studies; Cell Proliferation; Cells, Cultured; Conjunctiva; Epithelium; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; HLA-DR Antigens; Humans; Interferon-gamma; Interleukin-1; Interleukin-8; Keratins; Middle Aged; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; Sjogren's Syndrome; Statistics, Nonparametric; Transglutaminases; Up-Regulation

It has been suggested that human cultured gingival epithelial sheets may serve as a possible grafting material. The purpose of this study was to examine the biological characteristics of human cultured gingival epithelial sheets by epithelial differentiation and proliferation markers. Immunohistochemical localization of cytokeratin 19, involucrin and proliferating cell nuclear antigen (PCNA) were examined in human cultured gingival epithelial sheets samples from twenty patients. Cytokeratin 19-immunopositive cells were scattered mainly in the suprabasal layer. Immunoreactivity for involucrin was observed in all layers except for the basal layer. The majority of proliferating cell nuclear antigen-immunopositive cells was found in the basal layer. These results suggested that the cultured human gingival epithelial sheets were biologically active and in proliferative condition, which implies that this biological product may be a potential grafting material.

Topics: Adult; Aged; Biomarkers; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cells; Female; Gingiva; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Periodontitis; Proliferating Cell Nuclear Antigen; Protein Precursors

To determine the differentiation of human limbal epithelial cells in tissue culture.. Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR).. The colony-forming efficiency of primary cultured (P0) cells was about 19.35 +/- 6.46% (mean +/- SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P <0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 +/- 1.27)% (n = 7, P0) and (3.96 +/- 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse.. Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

Topics: Cell Differentiation; Coculture Techniques; Connexin 43; Cornea; Epithelial Cells; Humans; Immunohistochemistry; Keratins; Limbus Corneae; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Tissue Culture Techniques

Previously we documented that human epidermis exclusively expresses corticotropin releasing hormone receptor 1 (CRH-R1). To define the role of CRH in the epidermis, we investigated its effects on differentiation of normal human adult epidermal keratinocytes. Thus, CRH inhibited proliferation in a dose dependent fashion and significantly decreased Ki-67 antigen expression. This effect was independent of either the presence or the absence of growth factors in the medium. Flow cytometry analysis demonstrated that CRH inhibited the transition from G0/1 to S phase of the cell cycle, which was accompanied by an increased expression of cdk inhibitor p16 (Ink4a) protein. The antiproliferative effect was attenuated by protein kinase C inhibitor (GF109203X) but not by H89 (protein kinase A inhibitor), PD98059, or SB203580 (MAP kinase inhibitors). The cell cycle withdrawal was associated with the induction of keratinocyte differentiation. Thus, CRH stimulated the expression of cytokeratin 1 and involucrin, and inhibited cytokeratin 14 on both mRNA and protein levels. It also increased cell granularity and cell size. Furthermore, CRH induced signal transduction cascade that included stimulation of inositol 1,4,5-triphosphate, which was time and dose dependent. CRH also increased activator protein-1 DNA binding activity with JunD identified as the most important element. Thus, activation of CRH-R1 induces a non-random and sequential signal transduction cascade governing both keratinocyte differentiation and the inhibition of cell proliferation through G0/1 arrest. We propose that this program, triggered by CRH interaction with CRH-R1, includes induction of a transduction pathway involving the sequential activation of phospholipase C, protein kinase C, activator protein-1 (including Jun D), and p16.

Topics: Adult; Biomarkers; Cell Differentiation; Cell Division; Cells, Cultured; Corticotropin-Releasing Hormone; Epidermal Cells; Epidermis; Flow Cytometry; Humans; Inositol Phosphates; Keratin-14; Keratinocytes; Keratins; Protein Kinase Inhibitors; Protein Precursors; Receptors, Corticotropin-Releasing Hormone; RNA, Messenger; Transcription Factor AP-1

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Cell Adhesion; Cell Movement; Cell Shape; Cells, Cultured; Chlorocebus aethiops; COS Cells; Fibroblasts; Gene Expression Regulation; Keratin-15; Keratin-5; Keratinocytes; Keratins; Ki-67 Antigen; Mice; Mice, Transgenic; Microscopy, Confocal; Precipitin Tests; Protein Precursors; Proto-Oncogene Proteins c-raf; rho GTP-Binding Proteins; Signal Transduction; Time Factors; Wound Healing

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.

Topics: Adult; Aged; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biomarkers; Bromodeoxyuridine; Cell Adhesion; Cell Division; Cells, Cultured; Collagen Type IV; DNA-Binding Proteins; Epithelial Cells; Genes, Tumor Suppressor; Humans; Integrin beta1; Keratin-12; Keratins; Limbus Corneae; Middle Aged; Neoplasm Proteins; Phenotype; Phosphoproteins; Protein Precursors; RNA, Messenger; Stem Cells; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

Corticotropin-releasing hormone (CRH) is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These are mediated by CRH-R1alpha and represented by adenylate cyclase activation, Ca2+ influx, inhibition of cell proliferation and modifications in intracellular signal transduction by NF-kappaB.. To define CRH action in the cell cycle we investigated its effects on the differentiation programme using the HaCaT keratinocytes model.. HaCaT keratinocytes were incubated with CRH in Dulbecco's modified Eagles's medium (containing 1.8 mmol L(-1) calcium) or EpiLife (containing 0.06 mmol L(-1) calcium) medium. Cell proliferation was assessed with the MTT assay. Flow cytometry was used for the measurement of DNA content, cell size and granularity and the expression of cytokeratin 14, cytokeratin 1 and involucrin. The electrophoretic mobility shift assay was used to determine DNA binding activity by AP-1 transcription factor. Expression of cytokeratin 1 was also assessed with immunofluorescence microscopy.. CRH did produce inhibition of proliferation, which was dose-dependent; the shape of the inhibition curve was determined by the media calcium concentration. CRH action was pinpointed at inhibition of the G0/1 to the S phase transition of the cell cycle. CRH also increased AP-1 binding activity, cell granularity, cytokeratin 1 and involucrin expression, and inhibited cytokeratin 14 expression.. These results are consistent with CRH induction of the keratinocyte differentiation programme. Thus, the overall CRH cutaneous actions connote protective functions for the epidermis, that appear to include the triggering or acceleration of the differentiation programme.

Topics: Cell Differentiation; Cell Proliferation; Cell Size; Cells, Cultured; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Humans; Interphase; Keratinocytes; Keratins; Protein Precursors; Receptors, Corticotropin-Releasing Hormone; Transcription Factor AP-1

Cutaneous xerosis is a common clinical condition associated with an altered barrier function of the stratum corneum. Xerotic skin appears dry, rough and slightly scaling. Patients complain of pruritus and stinging. Our aim was to investigate the clinical effects of a cosmetic ointment (Scherilan) in patients with circumscribed senile xerosis (also called asteatotic eczema). Moreover, variations in expression of epidermal proteins such as keratin (K)-5 and involucrin, detected by immunohistochemistry, were also evaluated before and after topical treatment. We enrolled 30 patients (11 males, 19 females) with asteatotic eczema. We examined dryness, roughness and desquamation and symptoms such as itching and dryness. A score of 0 to 3 was assigned to each of these parameters. A biopsy was performed in seven patients before and after a 21-day topical treatment. All skin specimens were then immunostained with antibodies to K5 and involucrin. At day 7 or 21 of treatment all signs of xerosis and pruritus were significantly reduced; furthermore, the reduction increased with the duration of therapy. Before treatment K5 was strongly expressed in stratum basale (SB) and stratum spinosum (SS), while involucrin was strongly expressed in stratum granulosum (SG) and the upper portion of SS. In contrast, after treatment immunostaining for K5 was restricted to SB and the lower part of SS, while involucrin showed intense staining in SG. We highlight the importance of treating cutaneous xerosis with an ointment such as this one, which probably induces an increase of lipid content of the SC intercellular matrix.

Topics: Aged; Biopsy; Cell Proliferation; Cosmetics; Eczema; Epidermis; Female; Glycolates; Humans; Immunohistochemistry; Keratin-5; Keratins; Lipid Metabolism; Lipids; Male; Middle Aged; Ointments; Protein Precursors; Pruritus; Skin Diseases; Time Factors; Treatment Outcome; Vitamin E

Topics: Cell Differentiation; Female; Humans; Immunohistochemistry; Keratins; Ki-67 Antigen; Membrane Proteins; Middle Aged; Protein Precursors; Sjogren's Syndrome; Skin Diseases

Phenylketonuria (PKU) is a metabolic disease causing increased levels of phenylalanine in blood and body fluids. Circulating phenylalanine is normally cleared by phenylalanine hydroxylase (PAH) expressed in the liver. The aim of this study is to exploit the skin as a 'metabolic sink' removing phenylalanine from the blood. We have previously showed that the overexpression of PAH and GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in the synthesis of the cofactor for PAH, leads to high levels of phenylalanine clearance in primary human keratinocytes. In this study, we have investigated the 'metabolic sink' strategy in an in vivo model by developing three lines of transgenic mice expressing PAH and GTP-CH in various layers of the skin. The promoters used were keratin 14 (K14), involucrin (INV) and a truncated variant of Keratin 1 (K1). The mice were crossbred to a mouse model of human PKU, the PAH(enu2) mouse, in order to obtain mice that do not express PAH in the liver and the kidney. Transgenic mice containing the INV and K14 promoters expressed PAH and GTP-CH in the epidermis. However, the K1 promoter did not lead to detectable gene expression. Analysis of the mice showed that no phenotypic effect was observed in mice expressing PAH and GTP-CH from the INV promoter. However, low level of phenylalanine clearance was observed in mice expressing PAH and GTP-CH from the K14 promoter, suggesting that the skin can be genetically engineered to function as a 'metabolic sink'.

Topics: Animals; Base Sequence; Disease Models, Animal; DNA, Complementary; Gene Expression; Genetic Engineering; Genetic Therapy; GTP Cyclohydrolase; Humans; Keratin-14; Keratins; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Transgenic; Phenotype; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Promoter Regions, Genetic; Protein Precursors; Skin

Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m(3) or 80 mg/m(3), 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers.

Topics: Animals; Cell Proliferation; Cyclin D1; Enzyme Activation; Filaggrin Proteins; Hyperplasia; Intermediate Filament Proteins; JNK Mitogen-Activated Protein Kinases; Keratins; Lung Neoplasms; Male; Membrane Proteins; Metaplasia; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Inbred WKY; Signal Transduction; Smoking; Transcription Factor AP-1

Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

Topics: Animals; Animals, Newborn; Antibody Specificity; Calcitriol; Calcium; Calcium Channel Agonists; Carcinogens; Cell Differentiation; Cell Division; Chlorocebus aethiops; COS Cells; Extracellular Space; Keratin-15; Keratin-5; Keratinocytes; Keratins; Mice; Mice, Inbred ICR; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C; Protein Precursors; Tetradecanoylphorbol Acetate; Transglutaminases

To determine the role played by keratinocyte transglutaminase (TG1, TG(K)) in the abnormal keratinization of the cornea.. Vitamin A-deficient rats were produced as a model of severe dry eyes, and the expression of the mRNA and the enzyme activity of TG1 were examined in the corneas. The envelope proteins and keratins of cornified cells were also examined immunohistochemically.. The expression and enzyme activity of TG1 mRNA on the ocular surface were significantly upregulated as the vitamin A deficiency developed. As the TG1 expression was upregulated, involucrin, loricrin, and keratin 10 began to be expressed on the epithelial cells of the cornea.. Upregulation of TG1 expression followed by the appearance of the envelope proteins and keratin10 in cornified cells indicated that TG1 is involved in the abnormal keratinization of the cornea.

Topics: Animals; Blotting, Northern; Cornea; Disease Models, Animal; Dry Eye Syndromes; Epithelial Cells; Gene Expression Regulation, Enzymologic; Immunoenzyme Techniques; Keratin-10; Keratins; Membrane Proteins; Protein Precursors; Rats; Rats, Sprague-Dawley; Retinaldehyde; RNA, Messenger; Transglutaminases; Up-Regulation; Vitamin A; Vitamin A Deficiency

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.

Topics: Animals; Biolistics; Blotting, Northern; Blotting, Western; Capsid; Cell Differentiation; Cells, Cultured; Chromatography, High Pressure Liquid; Codon; Dactinomycin; DNA; Gene Expression Regulation, Viral; In Vitro Techniques; Keratin-14; Keratinocytes; Keratins; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Nucleic Acid Hybridization; Papillomaviridae; Plasmids; Protein Biosynthesis; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; RNA, Transfer; Time Factors; Transfection; Viruses

It has been suggested that human cultured gingival epithelial sheets may serve as a possible grafting material. The purpose of this study was to examine the biological characteristics of human cultured gingival epithelial sheets by epithelial differentiation and proliferation markers. Immunohistochemical localization of cytokeratin 19, involucrin and proliferating cell nuclear antigen (PCNA) were examined in human cultured gingival epithelial sheets samples from twenty patients. Cytokeratin 19-immunopositive cells were scattered mainly in the suprabasal layer. Immunoreactivity for involucrin was observed in all layers except for the basal layer. The majority of proliferating cell nuclear antigen-immunopositive cells was found in the basal layer. These results suggested that the cultured human gingival epithelial sheets were biologically active and in proliferative condition, which implies that this biological product may be a potential grafting material.

Topics: Adult; Aged; Biomarkers; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cells; Female; Gingiva; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Proliferating Cell Nuclear Antigen; Protein Precursors

Previously we demonstrated that genetic deficiency of the cyclooxygenases (COX-1 or COX-2) altered keratinocyte differentiation in mouse skin [Tiano et. al. (2002) Cancer Res. 62, 3395-3401]. In this study, we show that topical application of SC-560 (a COX-1 selective inhibitor) or celecoxib (COX-2 selective) to TPA-treated wild-type skin caused fivefold increases in the number of basal keratinocytes expressing the early differentiation marker keratin 1 (K1). In contrast to skin, COX-2 not COX-1 was the major isoform expressed in cultured primary keratinocytes. COX-1 was predominantly expressed in detached, differentiated cells, whereas COX-2 was found in the attached, proliferating cells. High Ca++ medium induced K1 and COX-1 in wild-type keratinocytes but did not change COX-2 expression. As observed in skin, COX-1-/- and COX-2-/- primary keratinocytes expressed fivefold more K1 than wild-type cells. K1 levels in cultured wild-type keratinocytes were also increased by treatment with celecoxib and indomethacin. However, unlike its in vivo effect, SC-560, possibly due to low COX-1 expression in cultured mouse keratinocytes, did not increase K1 levels. Furthermore, no increases in apoptotic cell numbers were observed in COX-deficient keratinocytes or COX-inhibitor treated wild-type cells. Thus, a major effect of COX inhibitors and COX-deficiency is the induction of keratinocyte differentiation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Celecoxib; Cell Differentiation; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Epidermal Cells; Isoenzymes; Keratinocytes; Keratins; Membrane Proteins; Mice; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Protein Precursors; Pyrazoles; Skin Neoplasms; Sulfonamides

Full-thickness excisional wounds were made on the dorsal skin of 1-day-old rats to elucidate from where the cells move into the defect and what kinds of cells they are. Immunohistochemical analyses of the wound sites revealed that the following two subsets of keratinocytes were the major contributors to reepithelialization: first, the cells at the forefront of the migrating epithelium, termed "leading edge cells," which expressed K14 keratin, known as basal cell-specific keratin, but not K6 or K10 keratins, so that they had probably moved from the basal cell layer; and, second, the cells tentatively termed "immature spinous cells," which expressed K14 and K6 but not K10, and formed an "ingrowth region" following the leading edge cells. These two kinds of cells moved to the open wound area, as a multilayered cell sheet. Fluorescent phalloidin staining experiments indicated that actin filaments became concentrated in the leading edge cells within 6 h postwounding (PW), whereas weak signals of actin filaments were detected in the immature spinous cells. Taken together, the present findings support the view that wound covering in neonatal rat skin is accomplished by a movement en masse of keratinocytes from the bottom half of the surrounding epidermis.

Topics: Actin Cytoskeleton; Animals; Epidermal Cells; Epidermis; Immunohistochemistry; Keratinocytes; Keratins; Protein Precursors; Rats; Rats, Sprague-Dawley; Wound Healing

The presence of characteristic epithelial swirls called Hassall bodies within the human thymic medulla has been used as an indicator of ongoing or recent thymopoiesis. We present a case where Hassall bodies were present in the absence of current or past thymopoiesis. The patient had been treated with corticosteroids for presumed asthma before his diagnosis of X-linked SCID. Two other cases of nonimmunodeficient patients treated with high-dose corticosteroids had markedly increased numbers of thymic Hassall bodies. To determine whether corticosteroid treatment induces thymic epithelial (TE) differentiation to form Hassall bodies, mAbs reactive with specific cytokeratins (CKs), filaggrin, and involucrin were used to define distinct stages of TE cell differentiation. Treatment of primary TE monolayers with hydrocortisone in vitro induced expression of involucrin and high-molecular-mass CKs that are characteristic of TE differentiation. Treatment of thymic organ cultures with hydrocortisone induced both medullary and subcapsular cortical TE cells to express CK6, a differentiation marker that is normally expressed only by Hassall bodies in vivo. These experimental studies combined with the case observations indicate that exogenous corticosteroids can regulate terminal differentiation of TE cells both in vitro and in vivo. Thus, the presence of Hassall bodies in thymus from corticosteroid-treated patients cannot be taken as an absolute indication of previous thymopoiesis. Because corticosteroids are also made within the thymus under normal physiologic conditions, these studies support the hypothesis that endogenous corticosteroids may play a role in normal TE differentiation and Hassall body formation in vivo.

Topics: Adrenal Cortex Hormones; Antibodies, Monoclonal; Cell Differentiation; Chromosomes, Human, X; Epithelial Cells; Female; Filaggrin Proteins; Humans; Hydrocortisone; Immunophenotyping; Infant; Infant, Newborn; Intermediate Filament Proteins; Keratins; Lymphopoiesis; Male; Organ Culture Techniques; Protein Precursors; Severe Combined Immunodeficiency; T-Lymphocytes; Thymus Gland

Topics: Carcinoma, Squamous Cell; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Keratoacanthoma; Protein Precursors; Skin Neoplasms

For ethical and technical reasons, the in vivo biological effects of ultraviolet (UV) radiation on skin are difficult to study in human volunteers. The use of human skin grafted on to nude mice may circumvent this difficulty.. To investigate the effects of a single moderate UVB exposure on human skin grafted on to nude mice.. Modifications of epidermal differentiation markers and patterns of keratin expression were assessed from 24 h to 14 days after a physiological UVB irradiation characterized by the induction of sunburn cells.. During the first 48 h postexposure, involucrin, loricrin, transglutaminase type I, filaggrin and keratin K2e expression were altered together with the formation of abnormal horny layers. Constitutive keratin K14 was increased while keratin K10 expression was delayed. Newly synthesized keratins K6, K16, K17 and K19 were induced in parallel with an increase in the epidermal proliferation rate. A progressive normalization of both keratinocyte proliferation and differentiation took place during the following days, reaching completion within 2 weeks.. Exposure of human skin to a UVB dose corresponding to a mild sunburn reaction induces epidermal hyperproliferation and alterations of several constitutive differentiation markers, as well as a drastic modification in the pattern of epidermal keratins. Although these modifications were shown to be progressively reversed in a single exposure model, the data also suggest that subsequent UV exposures occurring during the recovery period may lead to potentially deleterious long-term consequences, such as photoageing and photocarcinogenesis. Grafted human skin appeared to be an attractive and promising model for investigating the biological consequences of UVB radiation in vivo.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Division; Dose-Response Relationship, Radiation; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Membrane Proteins; Mice; Mice, Nude; Protein Precursors; Radiation Injuries; Skin; Skin Transplantation; Sunburn; Transglutaminases; Transplantation, Heterologous; Ultraviolet Rays

We previously demonstrated that the aspartate protease cathepsin D is activated by ceramide derived from acid sphingomyelinase. Increased expression of cathepsin D in the skin has been reported in wound healing, psoriasis and skin tumors. We explored specific functions of cathepsin D during epidermal differentiation. Protein expression and enzymatic activity of cathepsin D increased in differentiated keratinocytes in both stratified organotypic cultures and in mouse skin during epidermal barrier repair. Treatment of cultured keratinocytes with exogenous cathepsin D increased the activity of transglutaminase 1, known to cross-link the cornified envelope proteins involucrin and loricrin during epidermal differentiation. Inhibition of cathepsin D by pepstatin A suppressed the activity of transglutaminase 1. Cathepsin D-deficient mice revealed reduced transglutaminase 1 activity and reduced protein levels of the cornified envelope proteins involucrin and loricrin. Also, amount and distribution of cornified envelope proteins involucrin, loricrin, filaggrin, and of the keratins K1 and K5 were significantly altered in cathepsin D-deficient mice. Stratum corneum morphology in cathepsin D-deficient mice was impaired, with increased numbers of corneocyte layers and faint staining of the cornified envelope only, which is similar to the human skin disease lamellar ichthyosis. Our findings suggest a functional link between cathepsin D activation, transglutaminase 1 activity and protein expression of cornified envelope proteins during epidermal differentiation.

Topics: Animals; Cadaverine; Cathepsin D; Cell Differentiation; Cells, Cultured; Enzyme Activation; Filaggrin Proteins; Gene Deletion; Intermediate Filament Proteins; Keratinocytes; Keratins; Membrane Proteins; Mice; Pepstatins; Protein Precursors; Protein Processing, Post-Translational; Skin; Transglutaminases; Wound Healing

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.

Topics: Amino Acid Sequence; Animals; Base Sequence; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Keratins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Protein Precursors; Psoriasis; Recombinant Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Skin; Tissue Distribution

Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients.. To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site.. We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings.. Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal.. Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Topics: Adult; Aged; Cadherins; Cell Adhesion; Cell Differentiation; Cells, Cultured; Codon, Nonsense; Cytoskeletal Proteins; Desmoglein 1; Desmogleins; Desmoplakins; Desmosomes; Epidermis; Humans; Keratins; Keratoderma, Palmoplantar; Microscopy, Electron; Middle Aged; Protein Precursors

A defective permeability barrier leads to the penetration of environmental allergens into the skin and initiates immunological reactions and inflammation crucially involved in the pathogenesis of atopic dermatitis (AD). Decreased stratum corneum ceramide content may cause the defect in permeability barrier function consistently found in AD. Acid and neutral sphingomyelinase (A- and N-SMase) generate ceramides with structural and signal transduction functions in epidermal proliferation and differentiation. We determined epidermal SMase activities, DNA synthesis, involucrin, loricrin, filaggrin, and keratin expression in lesional and non-lesional skin of AD patients. We found decreased epidermal A-SMase activity in lesional and non-lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function. N-SMase activity was reduced in non-lesional skin and more significantly reduced in lesional skin, correlating with impaired expression of cornified envelope proteins and keratins, important for skin barrier function. Changes in involucrin, loricrin, filaggrin, keratin K 5 (basal) and K 16 (proliferation associated) were noticed in non-lesional and lesional skin, whereas changes in K 10 (suprabasal), K 6 (proliferation associated), and K 17 (inflammation associated) were found only in lesional skin. In summary, reduction in SMase-generating ceramides and impaired differentiation are involved in the defective barrier function found in AD.

Topics: Biomarkers; Blotting, Western; Cell Differentiation; Cell Division; Ceramides; Dermatitis, Atopic; Epidermis; Female; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratins; Male; Membrane Proteins; Protein Precursors; Signal Transduction; Sphingomyelin Phosphodiesterase; Substrate Specificity; Water

This study examines differences between cultures of normal human oral epithelial cells and two squamous cell carcinoma cell lines (SCC15 and SCC25) in the expression of structural proteins, adhesion molecules, plasma membrane lipid composition, and intercellular junctions. Based on immunocytochemistry, most normal cell cultures appeared to express more E-cadherin, integrin beta-1, cytokeratin (CK) 14, CK19, and involucrin than SCC cultures. By Western blot analysis, normal cultures expressing high levels of E-cadherin also expressed high levels of involucrin and low levels of CK19. Both SCC cultures demonstrated lower expression of E-cadherin and involucrin, whereas only SCC15 cells showed high levels of CK19. Expression of beta-catenin, an E-cadherin associated protein with potential oncogene function, did not vary among normal and SCC cells. Proportions of saturated fatty acids quantified by thin layer chromatography were higher in the normal cell cultures, than in both SCC cell lines. No morphological differences were evident by transmission electron microscopy (TEM) between normal and SCC cell-cell intercellular junctions. Although no quantitation was attempted, observation suggested that normal cells form more intercellular junctions (TEM observation) and larger intercellular bridges (SEM observation) compared to both SCC cell lines. Of the factors examined, main variations between cultures of normal oral epithelium and the two SCC cell lines examined include the expression of structural and adhesion proteins, lipid composition, and intercellular junctions. The extent of the differences varies according to the stage of terminal differentiation demonstrated by the normal cell cultures.

Topics: Biomarkers, Tumor; Blotting, Western; Cadherins; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Fatty Acids; Gingiva; Humans; Immunohistochemistry; Integrin beta1; Keratinocytes; Keratins; Microscopy, Electron; Mouth Neoplasms; Protein Precursors

Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-beta-cyclodextrin (MbetaCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MbetaCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MbetaCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MbetaCD treatment induces a prolonged phosphorylation of p38 in general and p38alpha in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MbetaCD, but not by a p38delta-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38alpha/beta.

Topics: Adult; beta-Cyclodextrins; Cell Differentiation; Cells, Cultured; Cholesterol; Cyclodextrins; Enzyme Activation; Epidermal Cells; ErbB Receptors; Gene Expression; Humans; Keratin-10; Keratin-14; Keratinocytes; Keratins; Mitogen-Activated Protein Kinase 11; Mitogen-Activated Protein Kinase 14; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Precursors; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Up-Regulation

Topics: Animals; Epidermis; Filaggrin Proteins; Intermediate Filament Proteins; Keratins; Mice; Mice, Transgenic; Protein Precursors

To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca(2+) concentration and serum in modulating their growth and differentiation.. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4 degrees C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca(2+) concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin.. Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca(2+) but suppressed by 5% FBS in KSFM at passage 0.. Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca(2+) concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca(2+) concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Topics: Animals; Calcium; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Survival; Connexin 43; Culture Media, Serum-Free; Epidermal Cells; Epithelial Cells; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Immunoblotting; Keratins; Limbus Corneae; Membrane Proteins; Mice; Phosphoproteins; Protein Precursors; Zonula Occludens-1 Protein

Loricrin is a major component of the epidermal cornified cell envelope, and is expressed only in terminally differentiated keratinocytes. This cell differentiation-specific expression pattern suggests specific suppression of loricrin gene expression in undifferentiated keratinocytes as well as its activation in differentiated keratinocytes. We identified a negative regulatory sequence element in the first intron of the mouse loricrin gene involved in suppression of loricrin gene expression in undifferentiated keratinocytes. A database search indicated that this sequence contained the putative inverted Yin-Yang 1 (YY1)-binding motif. Constructs with point mutations in the putative YY1-binding motif showed increased reporter activity, indicating that YY1 negatively regulates loricrin gene transcription. Co-transfection experiments using a YY1 expression vector revealed that YY1 represses loricrin promoter activity. Western blotting and immunohistochemical analyses indicated that YY1 is more abundant in undifferentiated than in differentiated keratinocytes. These findings suggest that YY1 contributes to specific loricrin gene expression in differentiated keratinocytes by suppression of its transcription in undifferentiated keratinocytes. Furthermore, we demonstrated that forced expression of YY1 in differentiated keratinocytes results in specific downregulation of expression of other early and late differentiation markers.

Topics: Animals; Biomarkers; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Down-Regulation; Erythroid-Specific DNA-Binding Factors; Gene Expression Regulation; Introns; Keratin-10; Keratinocytes; Keratins; Membrane Proteins; Mice; Promoter Regions, Genetic; Protein Precursors; Transcription Factors; Transcription, Genetic; YY1 Transcription Factor

Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis. Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions. We have used telomerase technology to establish normal human oesophageal cell lines.. Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells. Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector. The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12. Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure.. Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40-45. In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length. These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact.. By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers. This cell line may be useful for studying the early events in oesophageal carcinogenesis.

Topics: 3T3 Cells; Animals; Calcium; Cell Cycle; Cell Line; Coculture Techniques; Epithelial Cells; Esophagus; Genetic Vectors; Humans; Keratinocytes; Keratins; Mice; Protein Precursors; Retroviridae; Telomerase; Transduction, Genetic

It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous cell carcinoma components were enlarged and also positive for HPV. This is thought to indicate that after adenocarcinoma cells are infected with HPV, they undergo morphological changes, and that "squamous metaplasia" follows. In this present study, the effects of HPV transfection into adenocarcinoma cells were examined. The relation between the region expressing the HPV gene and squamous metaplasia was also studied.. Plasmid pBR322 containing HPV type 16 (HPV-16) was transfected into cultured colonic adenocarcinoma (DLD-1) and lung adenocarcinoma (PC-14) cells using the calcium phosphate method. Neomycin was used as a selection marker. The presence of HPV E1, E2, E4, E5, E6, E7, L1, and L2 mRNAs and also transglutaminase 1, involucrin, cyclin dependent kinases (CDKs), cyclins, caspases, apoptosis inducing factor, DNase gamma, Fas, and Fas ligand mRNAs in HPV transfected cells was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The G0-G1 cell population was analysed by flow cytometry. Morphological examination under light and electron microscopes was also carried out.. The virus transfected cells showed squamous metaplasia when they were injected into severe combined immunodeficient mice, expressing the high molecular weight keratin (Moll's number 1 keratin) and involucrin molecules immunohistochemically, and involucrin and transglutaminase I mRNAs by RT-PCR. The squamous metaplasia was most conspicuous in the HPV transfected DLD-1 cell when compared with HPV transfected PC-14 cells. Squamous metaplasia was most clearly demonstrated in one HPV transfected DLD-1 cell clone, which expressed not only E2 but also E6-E7 fusion gene mRNA. Viral L1 mRNA expression was absent in HPV transfected cell clones, and was not related to squamous metaplasia. The growth rate of HPV transfected cells was reduced. Transfection of the virus into the cultured adenocarcinoma cells increased the G0-G1 cell population greatly, as assessed by flow cytometer analysis. Furthermore, in the virus transfected cells, apoptosis was also observed by means of the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling method.. HPV transfection into adenocarcinoma cells induced clear squamous metaplasia. One of the HPV transfected cell clones that expressed E2 and E6-E7 fusion gene mRNA showed the squamous metaplasia particularly clearly, and apoptosis was also demonstrated.

Topics: Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Differentiation; DNA, Viral; Humans; Keratins; Lung Neoplasms; Metaplasia; Mice; Mice, SCID; Neoplasm Proteins; Neoplasm Transplantation; Papillomaviridae; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Viral; Transfection; Tumor Cells, Cultured

Progenitor cells within the prostate basal layer may play important roles in differentiation and carcinogenesis; however, prostate stem cell populations remain uncharacterized.. Immunohistochemical and immunoblot analyses were used to characterize prostate epithelial cells (PrEC), a commercially available prostate basal cell isolate.. Proliferating PrECs exhibited immunophenotypic characteristics most consistent with basal cells, but during senescence PrECs up-regulated androgen receptor (AR) mRNA, p27, and low-molecular-weight cytokeratin (LMWCK) expression, suggestive of partial differentiation. PrECs also stained strongly for involucrin, which marked a subset of intermediate prostate basal cells in vivo. Basal hyperplasia consisting of involucrin-positive cells was prevalent in prostate tissue from androgen-ablated patients, and formed epithelial clusters flanked by involucrin-negative basal and luminal monolayers. Cultivation of PrECs on matrigel together with androgen-treated stromal conditioned media resulted in dense aggregates, with a peripheral rim of basal-like cells expressing p63 and basal cytokeratins.. PrEC represents an epithelial population whose basal characteristics are modified in response to matrigel, stromal factors, and senescence, consistent with a transient amplifying population. These cells may derive from a previously unrecognized, involucrin-positive subset present in vivo.

Topics: Blotting, Western; Cell Differentiation; Cellular Senescence; Epithelial Cells; Humans; Immunohistochemistry; Keratins; Male; Prostate; Prostatic Neoplasms; Protein Precursors; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stem Cells

We report here that human esophageal keratinocyte stem cells are characterized by the expression of the low-affinity neurotrophin receptor p75(NTR) and differentially expressed cell adhesion molecules, the beta1 and beta4 integrins. The candidate stem cells could be fractionated from keratinocytes as a minor cell subset by means of immunocytochemical cell sorting based on the different levels of expression of these cell surface molecules. Flow cytometric analysis revealed that this minor cell subset retained a relatively slow-cycling phenotype in vitro. These cells expressed low levels of involucrin and cytokeratin 13, indicating that the p75(NTR)-positive cell subset is immature relative to the other predominant subpopulations coexpressing beta1 integrin at higher levels. The p75(NTR)-positive cell subset was crucial for achieving longevity and the greatest output of keratinocytes comprising all distinguishable subpopulations in vitro. This process was associated with self-renewal and self-amplification of the p75(NTR)-positive cell subset. These findings strongly implicate p75(NTR) as a stem cell marker, which will be valuable for prospectively investigating stem cell regulation in association with different biological processes including neoplastic transformation of regenerative epithelia.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Cell Cycle; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; Esophagus; Flow Cytometry; Humans; Immunohistochemistry; Integrin beta1; Integrin beta4; Keratinocytes; Keratins; Neoplasms; Phenotype; Propidium; Protein Precursors; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Subcellular Fractions; Time Factors

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling

Topics: Animals; Biological Evolution; Epidermis; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Marsupialia; Membrane Proteins; Monotremata; Protein Precursors; Rabbits; Rats

Keratin 10 (K10) is known to be tightly bound to the cornified cell envelope (CCE) and this binding is thought to play an important role in enhancing the structural integrity of the cornified cells. Bullous congenital ichthyosiform erythroderma (BCIE) is a genetic disorder of keratinization caused by gene mutations in the conserved sequences of keratin 1 (K1) or K10, which leads to abnormal suprabasal keratin network assembly. In BCIE patients' skin, the keratin network abnormalities make the upper spinous and granular keratinocytes fragile and result in blister formation. However, the exact pathomechanism of the hyperkeratosis seen in BCIE is still unknown. The involvement of the CCE in the pathomechanism of hyperkeratosis in BCIE is controversial. Abnormal CCE assembly may cause hyperkeratosis as reported in cases of lamellar ichthyosis. Binding of K10 to CCE is thought to be a vital connection between the suprabasal keratin filament network and CCE. We hypothesize that abnormal suprabasal keratin assembly caused by either K1 or K10 mutations can disrupt CCE formation, resulting in the hyperkeratosis observed in BCIE. To clarify whether K10 and keratin network defects affect CCE formation in vivo, the ultrastructural and immunohistological features of CCE were studied in the epidermis of two Japanese BCIE patients from two independent families carrying an identical missense mutation M150T in the helix initiation motif of K10. Ultrastructurally, a 15-nm-thick, dense, normal-appearing CCE was formed at the cell periphery of the keratinized epidermal cells. Light and electron microscopic immunolabeling revealed that the major CCE precursor proteins, involucrin and loricrin, were normally distributed and restricted to CCE of the epidermis. Immunofluorescent labeling showed that epidermal TGases, TGase 1, TGase 2 and TGase 3, were expressed normally in the epidermis. These findings suggest that a normal CCE is formed during the process of human epidermal keratinization, even if the suprabasal keratin filament network is disrupted as with this particular K10 mutation, M150T in BCIE.

Topics: Adult; Cell Membrane; Epidermis; Female; Filaggrin Proteins; GTP-Binding Proteins; Humans; Hyperkeratosis, Epidermolytic; Intermediate Filament Proteins; Keratin-10; Keratins; Male; Mutation, Missense; Protein Glutamine gamma Glutamyltransferase 2; Protein Precursors; Protein Structure, Secondary; Transglutaminases

Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density-induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKC alpha, beta, gamma, delta, epsilon, eta, theta, zeta), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the lat (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocyte-specific transglutaminase-1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down-regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density-induced differentiation of HaCaT cells.

Topics: Blotting, Western; Calcium; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Microscopy, Fluorescence; Protein Isoforms; Protein Kinase C; Protein Precursors; Tetradecanoylphorbol Acetate; Time Factors; Transglutaminases

In the literature, few studies based on clinical and histological evaluation analyze skin structural changes after transplantation to the oral cavity. Ten patients affected by squamous cell carcinoma of the oral cavity who were reconstructed with a free forearm flap were included in the current study to analyze skin alterations. The authors performed a histological and ultrastructural evaluation of skin samples from the free forearm flap before transplantation and 18 months after intraoral reconstruction. They analyzed cytokeratin and involucrin distribution, which represent markers of maturation and differentiation of epithelia. The aim of this study was to demonstrate whether skin "mucosalization" occurs. They found that the skin undergoes some morphological changes induced by the intraoral environment. Cytokeratin and involucrin distribution is mostly unchanged. This aspect is in favor of skin structure preservation. Thus, they found that "mucosalization" of the skin is not evident after 18 months.

Topics: Aged; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Male; Microscopy, Electron, Scanning; Middle Aged; Mouth; Mouth Neoplasms; Protein Precursors; Skin; Skin Transplantation; Surgical Flaps

The effects of UVB radiation on the proliferation and differentiation of epidermal keratinocytes were investigated with respect to timing, dosage, and repeated exposures.. Nine healthy volunteers were placed into three subgroups and exposed to UVB radiation on buttock skin using a Waldmann UV 800 unit fitted with Philips TL-20W/12 fluorescent lamps. Three volunteers were given 2 MED of UVB and biopsied at: pre-exposure, 24, 48 and 72 h after UVB exposure. For three volunteers, 1 MED, 2 MED, 3 MED of UVB were applied. After 48 h, biopsies were taken from non-irradiated and irradiated sites. Finally, three volunteers received 1 MED of UVB daily for 5 days, and the non-irradiated and irradiated sites were biopsied 48 h after the final exposure. The expression of proliferation and differentiation markers by keratinocytes were detected by immunohistochemical staining, and the results were analysed quantitatively by image analysis.. The expression of proliferation and differentiation markers was observed prominently 48 h after irradiation. Higher doses of UVB caused an increase in proliferation and differentiation marker expression. Repeated exposures potentiated the effect of UVB radiation.. UVB irradiation concomitantly promotes epidermal proliferation and differentiation. Responses were maximal 48 h after irradiation. This effect of UVB increases linearly according to dose and repetition.

Topics: Adult; Biopsy; Buttocks; Cell Division; Humans; Immunoenzyme Techniques; Keratinocytes; Keratins; Ki-67 Antigen; Male; Membrane Proteins; Protein Precursors; Skin; Ultraviolet Rays

Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.

Topics: Cell Division; Cell Movement; Cell Separation; Cells, Cultured; Dermis; Epidermal Cells; Epidermal Growth Factor; Epidermis; Fibroblasts; Humans; Immunohistochemistry; Integrin alpha3; Integrin alpha6; Integrin beta1; Integrin beta4; Keratin-10; Keratinocytes; Keratins; Morphogenesis; Platelet-Derived Growth Factor; Protein Precursors; Protein Subunits; Skin, Artificial; Transforming Growth Factor beta; Transglutaminases

Topics: Conjunctiva; Conjunctival Diseases; Humans; Immunohistochemistry; Keratins; Membrane Proteins; Metaplasia; Mucous Membrane; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transglutaminases

Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its alpha and beta subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel alpha subunit had been deleted. Newborn -/- mice, in which the alpha subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion.

Topics: Animals; Biopsy; Cell Differentiation; Epidermal Cells; Epidermis; Epithelial Sodium Channels; Gene Expression; Hyperplasia; Keratinocytes; Keratins; Lipid Metabolism; Mice; Mice, Knockout; Microscopy, Electron; Protein Precursors; Sodium; Sodium Channels

To investigate the effects of basement membrane proteins on the reconstruction of mucosa equivalent, oral mucosa substitute were cultured on (1) type I collagen gels, (2) type IV collagen-coated type I collagen gels, (3) laminin-coated type I collagen gels, and (4) type I collagen gels containing both type IV collagen and laminin. H/E and PAS staining showed that the characteristics of the oral mucosa were preserved under all the experimental conditions. However, the basal keratinocytes appeared cuboidal when the type I collagen gels were coated with type IV collagen plus laminin. The expression of the differentiation markers was similar, but weak staining of filaggrin, K13, and involucrin was observed with the type IV collagen plus laminin coating. Furthermore, electron microscopy revealed that the size of the basal keratinocytes was relatively small and uniform when both type IV collagen and laminin were used. These findings suggested that these two major basement membrane proteins are important in the process of differentiation in mucosal keratinocytes.

Topics: Animals; Basement Membrane; Biomarkers; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Collagen; Culture Techniques; Epithelial Cells; Filaggrin Proteins; Gels; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Laminin; Microscopy, Electron; Mouth Mucosa; Protein Isoforms; Protein Precursors; Rats; Tail

Tissue-specific promoters are becoming increasingly important in light of their effects on gene expression in gene therapy experiments. The regulation of gene expression may be as important as the delivery of the gene itself.. To determine the effects of the involucrin (INV), keratin 14 (K14) and cytomegalovirus (CMV) promoters on the expression of the reporter gene beta-galactosidase.. In vivo, plasmid DNA was introduced to BALB/c mice by gene gun. Skin biopsies were taken after 24 h for histology and beta-galactosidase staining. In tissue culture cells, plasmid DNA was introduced by transient transfection to cell lines 293 (transformed primary human embryonal kidney cells), NIH 3T3 (immortalized mouse fibroblasts) and human keratinocytes. Reporter gene expression was assayed by histochemical staining and chemiluminescence.. The K14 and INV promoter constructs showed beta-galactosidase gene expression only in the epidermis, while the CMV promoter showed gene expression in both the dermis and epidermis. In cell culture, the INV and K14 promoter constructs demonstrated significant beta-galactosidase expression in human keratinocytes, but minimal expression in 293 and NIH 3T3 cell types. CMV promoter constructs demonstrated significant expression in all cell types.. Gene expression can be regulated by different promoters both in vivo and in cell culture. Based on the physiological expression of the different promoters, gene expression can be restricted to certain cell types, tissues and skin layers.

Topics: Animals; beta-Galactosidase; Biolistics; Culture Techniques; Cytomegalovirus; Gene Expression Regulation; Genes, Reporter; Humans; Keratin-14; Keratinocytes; Keratins; Luminescent Measurements; Male; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; Protein Precursors; Skin; Transfection

Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice.. Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice.. In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells.. Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

Topics: Animals; Blotting, Northern; Blotting, Western; Chondroitin Sulfate Proteoglycans; Corneal Neovascularization; Corneal Stroma; Epithelium, Corneal; Eye Proteins; Fibroblasts; Filaggrin Proteins; Gene Expression; Hyperplasia; In Situ Hybridization; Intermediate Filament Proteins; Keratan Sulfate; Keratins; Lumican; Membrane Proteins; Mice; Mice, Mutant Strains; Protein Precursors; RNA, Messenger

The expression of cytokeratins and involucrin was analyzed to identify the skin cells which compose the epidermis of dogs. The distribution of cytokeratins and involucrin in normal dog skin was immunohistochemically examined with 27 commercial monoclonal antibodies for human use. Antibodies, No.4. OV-TL12/13, 35betaH11, 4.1.18, CAM5.2, NCL5D3, Ks.13.1, Ks.18.04, Ks.19.1, 170.2.]4 and Ks.20.8 stained hair follicles and/or the sweat gland duct, but not the epidermis. Antibodies, 34betaB4, AE3, 34betaE12. LP34, RCK102, MNF116, AE1, KLI, DE-K10 and DE-K13 reacted with every layer of the epidermis, hair follicles and the sweat gland duct. These results were similar to those reported in the human skin. No positive staining, however, could be detected in the epidermis, hair follicles and the sweat gland duct with commercial antibodies, 6B10, Ks.7.18, Mu146-uc, E3, RCK108 and involucrin. Therefore, immunohistochemical investigation with these commercial antibodies developed for human skin examination might be available for investigating the origin of skin tumors in dogs.

Topics: Animals; Antibodies, Monoclonal; Biopsy; Dogs; Epidermal Cells; Hair Follicle; Humans; Immunohistochemistry; Keratins; Protein Precursors; Reference Values; Skin; Sweat Glands

In severe ocular surface diseases, pathologic keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. The expression in conjunctivalized corneas of various proteins known to play important roles in the physiological keratinization process in human epidermis was examined to better understand the mechanism of keratinization.. Conjunctiva covering the cornea was examined in 12 eyes with ocular surface disease in the chronic cicatricial phase. These comprised four Stevens-Johnson syndrome, four ocular cicatricial pemphigoid, and four chemical injuries. Normal conjunctivas from four age-matched individuals served as controls. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate transglutaminase 1 gene expression and immunohistochemistry to study the expression of transglutaminase 1 protein along with other keratinization-related proteins (involucrin, loricrin, filaggrin, and cytokeratins 1 and 10) and cytokeratin pairs 4/13 and 3/12.. Semiquantitative RT-PCR showed that transglutaminase 1 mRNA expression was upregulated in keratinized conjunctiva compared with normal. Also, in this tissue, immunohistochemistry demonstrated elevated levels of transglutaminase 1, involucrin, filaggrin, and the cytokeratin pair 1/10. Levels of loricrin and cytokeratin pairs 4/13 and 3/12, however, remained the same.. Various keratinization-related proteins, transglutaminase 1 included, are most likely involved in the pathogenesis of cicatrizing ocular surface diseases.

Topics: Adult; Aged; Aged, 80 and over; Conjunctiva; Conjunctival Diseases; Female; Filaggrin Proteins; Fluorescent Antibody Technique, Indirect; Humans; Intermediate Filament Proteins; Keratins; Male; Membrane Proteins; Middle Aged; Pemphigoid, Benign Mucous Membrane; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stevens-Johnson Syndrome; Transglutaminases; Up-Regulation

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.

Topics: Animals; Antigens, Viral; Apoptosis; Bacterial Proteins; Cell Culture Techniques; Cell Line, Transformed; Clone Cells; Cytokines; Epithelial Cells; Female; Filaggrin Proteins; Gingiva; Immunohistochemistry; In Situ Nick-End Labeling; Intermediate Filament Proteins; Keratins; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; Simian virus 40

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Topics: Aging; Animals; Cattle; Cell Differentiation; Cell Division; Cyclooxygenase 2; Epidermis; Hair; Hair Follicle; Heterozygote; Hyperplasia; Isoenzymes; Keratins; Membrane Proteins; Mice; Mice, Transgenic; Phenotype; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; Sebaceous Glands; Skin

To investigate the pathophysiological role of matrix metalloproteinase (MMP)-9 in the skin, we analyzed MMP-9 expression from human keratinocytes in culture. MMP-9 and the terminal differentiation marker involucrin were co-localized in the same keratinocytes with a high concentration of Ca(2+), a potent stimulator of differentiation. We identified the novel KRE-M9 element, further downstream to the previously reported TPA responsive element in the MMP-9 promoter, and both of these two elements were shown to be important for MMP-9 transcription and Ca(2+) induction. The concomitant upregulation of MMP-9 and involucrin transcripts was probably due to the very similar gene regulatory elements, KRE-M9 and KRE-4, in their respective promoters. These results indicate a novel mechanism of transcriptional regulation for MMP-9 in the process of keratinization, implying the probable association of apoptosis and differentiation of keratinocytes in epidermal skin tissue.

Topics: Calcium; Cell Differentiation; Cells, Cultured; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Immunohistochemistry; In Situ Hybridization; Keratinocytes; Keratins; Matrix Metalloproteinase 9; Protein Precursors; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Skin

RHPS, composed of confluent allogeneic keratinocytes cultured on cell-free pig dermis, stimulates wound healing when applied with the keratinocyte layer facing the wound. So far it has not been clarified whether the confluent keratinocytes implanted 'upside-down' can 'take' or only stimulate healing by producing growth factors. Confluent male keratinocytes were grafted onto donor sites of three female patients. Biopsies were taken on days 4, 6 and 9 after grafting. The fate of donor cells was followed in paraffin sections by FISH for the Y chromosome and by persisting expression of vimentin taken as a marker of cultured keratinocytes. Histological evaluation was complemented by detection of keratin 10 and involucrin. All three donor sites healed within one week. On day 4 the early neoepidermis was multilayered but disordered after transplantation. A large proportion of cells were apparently of donor origin as indicated by the presence of Y chromosomes, irregular morphology, expression of vimentin in the bottom and upper layers of the neoepidermis, and by irregular expression of involucrin and keratin 10 only in the central layer of the neoepidermis. From day 6 onwards, the new epidermis acquired an ordered stratification. Involucrin and keratin 10 renewed normal distribution in suprabasal layers. Concomitantly, vimentin expression was decreasing. The Y chromosome was still found on day 6 but not on day 9. We concluded that confluent allogeneic keratinocytes temporarily 'take' to the wound and contribute to rapid wound closure, being replaced by the patient's epidermal cells after about one week.

Topics: Adult; Animals; Biomarkers; Burns; Cell Survival; Cells, Cultured; Coculture Techniques; Dermis; Female; Graft Survival; Humans; In Situ Hybridization, Fluorescence; Keratin-10; Keratinocytes; Keratins; Male; Protein Precursors; Swine; Tissue Engineering; Transplantation, Homologous; Transplants; Vimentin; Wound Healing; Y Chromosome

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), significantly induced IL-8 production and NF-kappaB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2.

Topics: Bacterial Proteins; Drosophila Proteins; Epithelial Cells; Fimbriae, Bacterial; Gingiva; Humans; Interleukin-8; Keratins; Lipopolysaccharide Receptors; Membrane Glycoproteins; NF-kappa B; Oligopeptides; Peptide Fragments; Protein Precursors; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.

Topics: DNA, Viral; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization; Keratin-14; Keratins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Protein Precursors; Transcription Factor AP-1; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

This work demonstrates that glycerol-preserved acellular allodermis can be used as support for the proliferation of human keratinocytes and that the characteristics of this bioengineered tissue suggest its possible use as a permanent skin substitute for therapeutic challenges such as extensive burns as well as its possible use as an in vitro model for pharmacological studies. The removal of all basal membrane components during preparation of the dermal support also provides an original in vitro situation that allows observation of the reorganization of the dermal-epidermal junction. The tissue composite obtained is constituted of dermis covered by a well attached, multistratified epithelium with morphological characteristics that resemble human epidermis as evidenced by light and transmission electron microscopy, including the neoformation, albeit incomplete, of the dermal-epidermal junction. Assessment of involucrin and cytokeratin 14 expression by immunohistochemical assays established differentiation patterns. Both immerse and air-liquid interface culture systems were tested.

Topics: Culture Techniques; Glycerol; Humans; Immunohistochemistry; Keratinocytes; Keratins; Protein Precursors; Skin, Artificial; Tissue Engineering

A case of the Voerner type palmoplantar keratoderma was studied for abnormalities of keratinization parameters. An enzyme and materials used to build the marginal band or cellular envelope of the cornified cell were all abnormally expressed; i.e. transglutaminase I (TGK), loricrin, and involucrin were abnormally immunostained. In the normal controls, their expression was limited to the upper epidermis, mainly in the granular layer. In the lesional skin, they were detected from the suprabasal layer to the lower horny layer. Filaggrin, the protein of the keratohyalin granule, was also expressed more widely than in controls. Ultrastructural abnormalities included a significantly higher frequency of composite keratohyalin granules than controls, early formation of a marginal band in the midepidermis, and, most remarkably, the clumping of tonofilaments causing vacuolization of the cytoplasm of affected keratinocytes.

Topics: Adult; Female; Filaggrin Proteins; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Keratoderma, Palmoplantar; Membrane Proteins; Protein Precursors; Skin; Transglutaminases

Different epithelia show extensive variation in differentiation. Epidermis and epithelium from the hard palate are both typical examples of orthokeratinized epithelia whereas buccal mucosa is an example of a non-keratinized epithelium. Each of these tissues can be distinguished morphologically and also by the expression of a number of structural proteins. Tissue explants derived from epidermis, hard palate or buccal mucosa were cultured at the air-liquid interface on collagen gels containing human dermal fibroblasts. Reconstructed epithelia that retained many of the morphological and immunohistochemical characteristics of the original tissue were formed. Cultures derived from epidermis and the hard palate both had a well-defined stratum basale, stratum spinosum, stratum granulosum and stratum corneum whereas cultures derived from buccal mucosa had no stratum granulosum or corneum and the cells retained their nuclei. Significantly more living cell layers were observed in both types of epithelia obtained from the mouth than in epidermis. The specific localization of proliferation and differentiation markers (Ki67, loricrin, involucrin, SPRR2, SPRR3 and keratin 10) closely resembled that of the tissue from which the cultures were derived. As identical three-dimensional culture models were used here, it is concluded that the differences observed between these epithelia were due to intrinsic properties of the keratinocytes.

Topics: Biomarkers; Cell Differentiation; Cell Division; Cell Nucleus; Cells, Cultured; Collagen; Cornified Envelope Proline-Rich Proteins; Culture Media; Epidermal Cells; Epithelial Cells; Fibroblasts; Gels; Gene Expression Regulation; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratinocytes; Keratins; Ki-67 Antigen; Membrane Proteins; Mouth Mucosa; Palate; Peptides; Proline-Rich Protein Domains; Protein Precursors; Proteins; Skin

The biological effects of 1,25-dihydroxyvitamin D3 are mediated by a nuclear receptor, the vitamin D receptor (VDR). Targeted ablation of the VDR in mice results in hypocalcemia, hypophosphatemia, hyperparathyroidism, rickets, osteomalacia, and alopecia. Normalization of mineral ion homeostasis prevents these abnormalities with the exception of the alopecia. Because 1,25(OH)2D3 has been shown to play a role in keratinocyte proliferation and differentiation, we undertook studies in primary keratinocytes and skin isolated from VDR null mice to determine if a keratinocyte abnormality could explain the alopecia observed. The basal proliferation rate of the VDR null and wild-type keratinocytes was identical both under proliferating and differentiating conditions. Assessment of in vivo keratinocyte proliferation at 4 days of age confirmed that VDR ablation did not have a significant effect. There was no difference in the basal expression of markers of keratinocyte differentiation (keratin 1, involucrin, and loricrin) in the keratinocytes isolated from VDR-ablated mice when compared with those isolated from control littermates. Similarly, in vivo expression of these genes was not altered at 4 days of age. When anagen was induced by depilation at 18 days of age, the VDR null mice had a profound impairment in initiation of the hair cycle. These data suggest that the alopecia in the VDR null mice is not attributable to an intrinsic defect in keratinocyte proliferation or differentiation, but rather to an abnormality in initiation of the hair cycle.

Topics: Alopecia; Animals; Calcitriol; Calcium; Cell Differentiation; Cell Division; Cells, Cultured; Keratinocytes; Keratins; Membrane Proteins; Mice; Mice, Hairless; Mice, Knockout; Mutation; Protein Precursors; Receptors, Calcitriol

Solanum glaucophyllum (Sg) (synonym S. malacoxylon) is a plant toxic to cattle due to its high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives. Sg causes a disease characterized by wasting and calcification of soft tissues. The effects of vitamin D are not only important in calcium homeostasis, but also in immune regulation, cell growth and cell differentiation. Skin samples in Sg-intoxicated and control heifers were studied histologically. Cellular differentiation and proliferation were analysed by immunohistochemical expression of cytokeratins, involucrin and proliferating cell nuclear antigen (PCNA). The results were obtained by image processing and analysis and were statistically evaluated. Sg-intoxicated cattle showed atrophy of epidermis and severe involution of hair follicles and of sebaceous and sweat glands. As judged by PCNA expression, cellular proliferation was reduced, even though the reduction was not statistically significant. The analysed markers of differentiation, e.g. involucrin and cytokeratins 10 and 11, changed in relation to Sg-poisoning. The possible pathogenesis of the skin lesions is discussed.

Topics: Animals; Antibodies, Monoclonal; Argentina; Body Weight; Cattle; Cattle Diseases; Cell Differentiation; Cell Division; Female; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Plant Poisoning; Proliferating Cell Nuclear Antigen; Protein Precursors; Skin Diseases; Solanaceae; Solanaceous Alkaloids; Vitamin D

The morphologic distinction between papillary and follicular neoplasms of the thyroid gland can be difficult, especially on small biopsy specimens or in fine-needle aspirations. To determine whether immunohistochemistry could help in achieving the correct diagnosis, we characterized the staining pattern for a series of papillary and follicular neoplasms of the thyroid gland. A pilot study was performed using a panel of antibodies, including high-molecular-weight keratin (HMWK, 34 beta E12), cytokeratin (CK) 5/6, CK7, CK13, CK14, CK20, AE1/AE3, CAM5.2, involucrin, and villin. Of these antibodies, involucrin and HMWK showed strong differential staining between follicular and papillary neoplasms. HMWK stained 91% of papillary carcinomas, including follicular variants, with a median of 53% positive cells, and involucrin stained 72.5% of papillary neoplasms with a median of 45% positive cells. HMWK stained only 20% of follicular neoplasms, whereas involucrin stained 29% of cases. Papillary neoplasms showed strong, although patchy, staining with HMWK and involucrin, whereas those follicular neoplasms that did have staining showed a weak, diffuse pattern of staining. We believe that HMWK, and involucrin to a lesser degree, could be useful in differentiating papillary from follicular neoplasms, especially for cytologic cell block material or for cases in which the architectural pattern is follicular.

Topics: Adenocarcinoma, Follicular; Biomarkers, Tumor; Carcinoma, Papillary; Carcinoma, Papillary, Follicular; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Protein Precursors; Thyroid Neoplasms

The cornified cell envelope (CCE) is an insoluble matrix of covalently linked proteins assembled in differentiating keratinocytes, providing a barrier against external insults. CCEs derived from HPV 11-infected tissue are fragile compared to those derived from healthy epithelium. To study a possible role for the E1()E4 protein, HPV 11-infected epithelium was examined for the distribution of this protein and three CCE proteins. CCEs were then purified from genital epithelium, fragmented, washed to remove nonassociated proteins, and analyzed for E1()E4 protein. In HPV 11-infected tissue, the E1()E4 protein was detected in the region of the CCE in differentiated keratinocytes. Loricrin and cytokeratin 10 (K10) were absent in E1()E4-positive cells, and E1()E4 protein was not detected in cells containing these proteins. E1()E4 protein was detected in immunoblots as a 10- to 11-kDa doublet in extracts of intact CCEs from infected tissue and in extracts of CCE fragments prepared without using reducing agents. Extraction with reducing agents eliminated E1()E4 detection, suggesting that disulfide bonding was involved in the association with CCE fragments. In addition, cyanogen bromide degradation experiments, immunofluorescence, and immunoelectron microscopy provided evidence that E1()E4 protein was associated with CCE fragments by covalent bonds other than disulfide bonds. We conclude that E1()E4 protein expression is associated with profound alterations in detection of loricrin and K10 in HPV 11-infected genital epithelium. The E1()E4 protein copurified with CCEs derived from infected epithelium and could be identified in CCE fragments, suggesting a possible role for E1()E4 in the development of CCE abnormalities.

Topics: Blotting, Western; Cells, Cultured; Condylomata Acuminata; DNA-Binding Proteins; Epithelium; Genital Diseases, Male; Genitalia, Male; Humans; Immunohistochemistry; Keratins; Male; Membrane Proteins; Microscopy, Immunoelectron; Oncogene Proteins, Viral; Papillomaviridae; Protein Precursors; Viral Proteins

Transglutaminase 1 (TGase 1) is a Ca(2+)-dependent enzyme which catalyzes epsilon-(gamma-glutamyl)lysine cross-linking of substrate proteins such as involucrin and loricrin to generate the cornified envelope at the cell periphery of the stratum corneum. We have shown that disruption of the TGase 1 gene in mice results in neonatal lethality, absence of the cornified envelope, and impaired skin barrier function. Based on the importance of TGase 1 in epidermal morphogenesis, we have now assessed its role in wound healing. In neonatal mouse skin, TGase 1 mRNA as well as keratin 6alpha was induced in the epidermis at the wound edges as early as 2 hours after injury and that expression continued in the migrating epidermis until completion of re-epithelialization. The TGase 1 enzyme co-localized on the plasma membrane of migrating keratinocytes with involucrin, but not with loricrin, which suggests the premature assembly of the cornified envelope. Similar injuries to TGase 1 knockout mouse skins grafted on athymic nude mice showed substantial delays in wound healing concomitant with sustained K6alpha mRNA induction. From these results, we suggest that activation of the TGase 1gene is essential for facilitated repair of skin injury.

Topics: Animals; Gene Expression Regulation; Keratins; Mice; Mice, Inbred Strains; Mice, Knockout; Mice, Nude; Protein Precursors; RNA, Messenger; Skin; Skin Transplantation; Transglutaminases; Wound Healing; Wounds, Penetrating

Keratinization of the ocular surface epithelium is associated with various disorders impairing vision. We immunohistochemically determined whether the ocular surface epithelia express involucrin, and whether its expression pattern may differ in benign vs. malignant disorders. Expression of cytokeratins was also examined to provide further information relative to the epithelial differentiation.. We evaluated 17 specimens; 6 specimens of the normal ocular surface epithelia, 3 specimens from cases of conjunctival intraepithelial neoplasia (CIN), 6 of conjunctival squamous cell carcinoma (SCC) and 2 of conjunctivae from cases of superior limbic keratoconjunctivitis (SLK).. Corneal epithelium exhibited intracellular immunoreactivity for involucrin. Four of the 6 specimens of bulbar conjunctival epithelium showed involucrin immunoreactivity in the perimembranous region, whereas the fornical conjunctiva was negative. Cornified envelope in SLK specimens was positive for involucrin. The CIN showed its immunoreactivity in the perimembranous region in all levels of the hyperproliferative epithelium without keratinization, i.e., similar to the bulbar conjunctiva. The neoplastic cells of well-differentiated SCC showed involucrin in the perimembranous region, and those of moderately- to poorly-differentiated SCC have involucrin in their cytoplasm. The expression pattern of cytokeratins was unrelated to grade of malignancy in ocular SCC.. The epithelia of normal subjects and of CIN expresses involucrin without keratinization. In contrary, the keratinized SLK epithelium markedly expresses involucrin in the cornified envelope. The subcellular immunolocalization of involucrin in the ocular SCC may help in evaluating the differentiation, i.e., malignancy, of neoplastic cells.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Carcinoma, Squamous Cell; Conjunctiva; Conjunctival Diseases; Conjunctival Neoplasms; Epithelium; Eye Proteins; Female; Filaggrin Proteins; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Keratoconjunctivitis; Male; Middle Aged; Protein Precursors

Keratin 14 (K14) is believed to play a pivotal role in the maintenance of epidermal cell shape and contributing to their resistance to mechanical trauma, thereby protecting the cells from lysing. Mice harboring a K14 null mutation produce phenotypic characteristics of epidermolysis bullosa simplex, a skin blistering disease (Lloyd et al., 1995, J Cell Biol 129:1329-1344). K14 null animals die several days after birth, making the detailed study of the consequences of K14 deletion in epidermal cell physiology in vivo particularly difficult. To define the consequences of K14 loss more precisely, we used an in vitro approach by isolating K14-/- cell lines and studying epidermal differentiation in the K14 null background. Several keratinocyte cell lines were generated from 6-day-old mice homozygous for a targeted disruption of the K14 gene (lines designated MKC-5, MKC-23, and MKC-33) and from their wild-type littermates (lines designated MKC-1 and MKC-6). Under low Ca2+ (0.066 mM) and low serum (2%) conditions, both wild-type and mutant cells were able to adhere to collagen type I-coated dishes and form epithelial sheets. They maintained basal epidermal cell characteristics and continued to proliferate without obvious signs of terminal differentiation; however, K14-/- cells proliferated two- to threefold slower than did their wild-type counterparts. The distribution of K5, the natural partner of K14, at the immunofluorescence level was also normal looking in the K14-/- MKC-5 cells, but with fewer filaments detectable, consistent with the approximately 20% reduction in K5 detectable on immunoblots. K17 expression was increased approximately 40% in the K14-/- cells. The levels of K15 and K16 were not different in the MKC-5 and MKC-6 cell lines, suggesting that they are not contributing factors to the stabilization of K5 in the mutant cells. K8, K19, and vimentin were undetectable in both lines. Both MKC-5 and MKC-6 cells underwent morphological and biochemical differentiation in response to a switch to high Ca2+ medium. These findings indicate that K14-/- MKC-5 cells preserve the morphological, biochemical, and physiological characteristics of epidermal cells for an extensive period of time in vitro, likely due to the compensatory expression of K17. The culturing capacity of these cells also permits the analysis of keratinocyte growth and differentiation in the absence of K14. In addition, the culturing methods we describe will be useful for the generation of ep

Topics: Animals; Calcium; Cell Adhesion; Cell Communication; Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; Cell Separation; Epidermal Cells; Keratin-14; Keratinocytes; Keratins; Mice; Mice, Knockout; Protein Precursors; Stem Cells

The immunophenotypes, especially expression of cytokeratins, in 13 cases of trichogenic tumors were examined to investigate their histogenesis. Four cases of multiple trichoepithelioma, five cases of classical solitary trichoepithelioma, one case of desmoplastic trichoepithelioma, one case of trichogenic trichoblastoma, one case of trichoblastic fibroma, and one case of giant solitary trichoepithelioma were retrieved. The immunoreactivities of the epithelial nests and the keratinous cysts in these tumors were similar to those of the outer root sheath and the infundibulum of normal hair follicles, respectively. From the comparative studies of the immunophenotypes with those of normal hair follicles, we speculated that all trichogenic tumors differentiate mainly toward the outermost layer of the outer root sheath between the lower part of the permanent portion and the upper part of the transient portion and some parts of them differentiate toward various other parts of the follicles. Although differentiation toward the other follicular structures can vary from case to case, there is no particular staining pattern specific for each kind of trichogenic tumor and no significant differences in immunoreactivity among them. Our observations support a recent notion that all neoplasms of follicular germinative cells should be grouped as a single entity.

Topics: Adult; Aged; Biomarkers, Tumor; Child; Female; Hair Follicle; Humans; Immunoenzyme Techniques; Immunophenotyping; Keratins; Male; Middle Aged; Neoplasms, Basal Cell; Protein Precursors; Skin Neoplasms

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPbeta within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPbeta in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPbeta-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPbeta-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPbeta-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPbeta plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

Topics: Animals; Calcium Signaling; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Cells, Cultured; DNA-Binding Proteins; Epidermal Cells; Keratin-10; Keratinocytes; Keratins; Membrane Proteins; Mice; Mice, Mutant Strains; Nuclear Proteins; Protein Precursors; Transcriptional Activation

Autocrine growth of human epidermal keratinocytes is initiated in subconfluent cell cultures in the absence of exogenous growth factors, at low calcium concentration of the medium and with sufficient cell density. Culture confluence inhibits keratinocyte proliferation and upregulates expression of early, keratin 10 (K10), and late, involucrin, markers of differentiation. In this report, the phenotype of autocrine keratinocytes was studied at high cell density (postconfluence), specifically after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), or all-trans retinoic acid (RA). At postconfluence, K10 is decreased but not involucrin. TPA upregulates involucrin expression, but not K10 in subconfluent keratinocytes. Treatment of confluent keratinocytes with RA downregulates K10, but upregulates involucrin. This in vitro culture model, unlike others, simulates for the first time the in vivo effects of RA, a member of the retinoid family which potently modulates keratinocyte differentiation and the expression of selected gene products. It thus can be developed to further examine epidermal differentiation.

Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Epidermal Cells; Humans; Keratin-10; Keratinocytes; Keratins; Protein Precursors; Tetradecanoylphorbol Acetate; Tretinoin; Up-Regulation

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

Topics: Cell Differentiation; Cell Line; Cell Membrane; Cornified Envelope Proline-Rich Proteins; Cytoskeletal Proteins; Desmoplakins; Desmosomes; Epithelium; Epitopes; Humans; Immunohistochemistry; Keratinocytes; Keratins; Membrane Proteins; Microscopy, Immunoelectron; Plakins; Protein Precursors; Proteins

The bcl-2 proto-oncogene is a known inhibitor of apoptosis; in normal human stratified squamous epithelium, its expression is restricted to the basal cell layer. To investigate the functional role of bcl-2 protein in the process of differentiation of oral keratinocytes, bcl-2 expression vector was transfected into SCC-25 cells, which normally undergo squamous cell differentiation in vitro while expressing specific differentiation markers, e.g., keratin 10/11 and involucrin. In bcl-2 transfected SCC-25 cells, the expression of these differentiation markers was markedly suppressed. The bcl-2 proto-oncogene may play a critical role in opposing the commitment to terminal differentiation and apoptosis of oral keratinocytes.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; DNA Fragmentation; Down-Regulation; Genes, bcl-2; Humans; Immunoenzyme Techniques; Keratinocytes; Keratins; Mouth Neoplasms; Protein Precursors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

In stratifying squamous epithelia, the cornified cell envelope (CE), a peripheral layer of crosslinked protein, is assembled sequentially from precursor proteins initially dispersed in the cytoplasm. Its major component is loricrin (37 kDa in mouse), which contributes from approx. 60% to >80% of the protein mass in different tissues. Despite its importance to the mechanical resilience and impenetrability of these tissues, detailed information has not been obtained on CE structure, even on such basic properties as its thickness or uniformity across a given CE or from tissue to tissue. To address this issue, we have studied CEs isolated from three murine epithelia, namely epidermis, forestomach and footpad, by electron microscopy of metal-shadowed specimens and scanning transmission electron microscopy (STEM) of unstained specimens. The former data reveal that the cytoplasmic surface is smoothly textured whereas the extracellular surface is corrugated, and that the average thickness is 15.3+/-1.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.0+/-0.8 kDa/nm2, which were remarkably consistent over all three tissues. These data imply that the mature CE has a uniquely defined thickness. To explain its uniformity, we postulate that loricrin forms a molecular monolayer, not a variable number of multiple layers. In this scenario, the packing density is one loricrin monomer per 7 nm2, and loricrin should have an elongated shape, 2.5-3.0 nm wide by approx. 11 nm long. Moreover, we anticipate that any inter-tissue variations in the mechanical properties of CEs should depend more on protein composition and cross-linking pattern than on the thickness of the protein layer deposited.

Topics: Animals; Animals, Newborn; Cross-Linking Reagents; Cystatins; Epithelial Cells; Freeze Fracturing; Keratins; Membrane Proteins; Mice; Mice, Inbred BALB C; Microscopy, Electron; Microscopy, Electron, Scanning Transmission; Protein Precursors; Proteinase Inhibitory Proteins, Secretory; Proteins; Serine Proteinase Inhibitors; Skin; Stomach; Transglutaminases

The life cycle of the papillomaviruses is closely linked to host cell differentiation, as demonstrated by the fact that amplification of viral DNA and transcription of late genes occur only in the suprabasal cells of a differentiated epithelium. Previous studies examining the pathogenesis of papillomavirus infections have relied on the use of organotypic raft cultures or lesions from patients to examine these differentiation-dependent viral activities. In this study, we used a simple system for epithelial differentiation to study human papillomavirus (HPV) late functions. We demonstrate that the suspension of HPV-infected keratinocytes in semisolid medium containing 1.6% methylcellulose for 24 h was sufficient for the activation of the late promoter, transcription of late genes, and amplification of viral DNA. These activities were shown to be linked to and coincide with cellular differentiation. Expression of the late protein E1(wedge)E4 and amplification of viral DNA were detected in the identical set of cells after suspension in methylcellulose. This technique was also used to analyze the differentiation properties of the cells which expressed the late protein E1(wedge)E4. While induction of the spinous layer markers involucrin and transglutaminase was compatible with late promoter induction, expression of the differentiation-specific keratin-10 was shown not to be required for HPV late functions. Interestingly, while the majority of normal human keratinocytes induced filaggrin expression by 24 h, this marker of the granular layer was induced in a smaller subset of HPV type 31 (HPV-31)-positive cells at this time point. The HPV-31-positive cells which expressed filaggrin did not induce the late protein E1(wedge)E4. Use of the methylcellulose system to induce epithelial differentiation coupled with the ability to perform a genetic analysis of HPV functions by using transfection of cloned viral DNA will facilitate the study of the regulation of the papillomavirus life cycle.

Topics: Cells, Cultured; Culture Media; Filaggrin Proteins; Gene Expression Regulation, Viral; Genome, Viral; Humans; Keratin-10; Keratinocytes; Keratins; Papillomaviridae; Protein Precursors; Transglutaminases; Viral Proteins; Virus Replication

A well-characterized collagen-glycosaminoglycan matrix (CGM) that has been shown to function as a dermal analog was seeded with freshly disaggregated autologous keratinocytes and applied to full-thickness wounds in a porcine model. CGM were impregnated with 50,000 keratinocytes per cm2, a seeding density that produces a confluent epidermis within 19 d post-grafting and affords a 60-fold surface expansion of the donor epidermis. In this study, the temporal sequence of events in epidermal and neodermal formation was analyzed histopathologically and immunohistochemically from 4 to 35 d post-grafting. The epidermis was observed to form from clonal growth of individual keratinocytes into epithelial cords and islands that gradually enlarged, coalesced, differentiated to form large horn cysts, and finally reorganized at the graft surface to form a fully differentiated, normally oriented epidermis with rete ridges. Simultaneously, a neodermis formed from migration of endothelial cells, fibroblasts, and macrophages into the CGM from the underlying wound bed, resulting in formation of blood vessels, the production of abundant extracellular matrix, and the degradation of the CGM fibers, respectively. Gradually, the stromal cellularity of the CGM decreased and collagen deposition and remodeling increased to form a neodermal connective tissue matrix beneath the newly formed epidermis. Complete dissolution of the CGM occurred, partly as a result of degradation by an ongoing foreign-body giant cell reaction that peaked at 8-12 d post-grafting, but neither acute inflammation nor evidence of immune stimulation were observed. Within 1 mo, many structural components of normal skin were reconstituted.

Topics: Animals; Antigens, Surface; Collagen; Factor VIII; Female; Glycosaminoglycans; Immunohistochemistry; Integrin alpha6beta4; Integrins; Keratinocytes; Keratins; Ki-67 Antigen; Laminin; Protein Precursors; Regeneration; Skin; Skin Physiological Phenomena; Skin Transplantation; Skin, Artificial; Swine; Time Factors; Transplantation, Autologous

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the epidermis, where it is thought to play a role in the regulation of keratinocyte growth and differentiation. Most in vitro studies of normal keratinocytes use monolayer cell cultures, which have limitations, including the inability to reproduce the stratified structure of the epidermis. The objective of this study was to investigate PTHrP production and secretion, and mRNA expression in skin organotypic cultures. The cultures consisted of an artificial dermis with differentiating keratinocytes grown at the air-liquid interface. Immunohistochemical assessment of cytokeratins 14 and 10/13, involucrin, and proliferative cell nuclear antigen (PCNA) demonstrated that keratinocytes differentiated in a manner similar to keratinocytes in normal epidermis. PTHrP expression was demonstrated in all viable layers of the epidermis, as well as in some fibroblasts of the collagen lattice by immunohistochemistry and in situ hybridization. Since most fibroblasts expressed alpha-smooth muscle actin, these cells were interpreted to be consistent with myofibroblasts. PTHrP expression by myofibroblasts suggests a possible role for PTHrP in the regulation of contractibility of these cells. PTHrP was also detected in conditioned media for 50 days. In conclusion, because of its superior tissue morphology and ability to induce organized keratinocyte differentiation, this culture system will be an excellent model to study the role of PTHrP in pathologic and physiologic processes involving the epidermis in vitro.

Topics: Animals; Cell Differentiation; Culture Media, Conditioned; Epidermis; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Keratinocytes; Keratins; Male; Organ Culture Techniques; Parathyroid Hormone-Related Protein; Proliferating Cell Nuclear Antigen; Protein Biosynthesis; Protein Precursors; Proteins; Rats; RNA, Messenger; Skin

The chemotherapeutic agent and vitamin A metabolite retinoic acid (RA) has been used to treat many tumor types. The effects of RA are mediated by a family of ligand-dependent transcription factors, the RA receptors and the retinoid X receptors (RXR). Alterations in retinoid receptor expression have been implicated in tumorigenesis. Previous studies have shown lack of RXR-gamma expression in squamous cell carcinoma (SCC) lines. To begin to elucidate the role of RXR-gamma in the malignant transformation of SCCs, we expressed RXR-gamma in SCC lines by stable transfection. SCC lines expressing RXR-gamma produced large numbers of flat cells with abundant cytoplasm, which died and detached from the culture dish. These cells morphologically resembled the differentiated cells of normal stratified squamous epithelium in culture. These cells did not exhibit the characteristic DNA fragmentation pattern of apoptotic cells, nor did they label in a fluorescent apoptosis assay. RNase protection and Western blot analysis revealed induction of RA-responsive involucrin and keratin 10 expression, early markers of terminal differentiation. RXR-gamma expression produced significant reduction in levels of RA-responsive genes including the cyclin-dependent kinase inhibitors p21Cip1/WAF1 and p27Kip1, resulting in increased cdc2 and cdk2 kinase activity and RB phosphorylation. We concluded that RXR-gamma induced terminal differentiation in SCC lines, suggesting a potential tumor suppressor function for this transcription factor.

Topics: Blotting, Western; Carcinoma, Squamous Cell; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Size; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Enzyme Activation; Genes, Tumor Suppressor; Humans; Keratin-10; Keratins; Microtubule-Associated Proteins; Phosphorylation; Precipitin Tests; Protein Precursors; Protein Serine-Threonine Kinases; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

In previous studies we have shown that experimental permeability barrier disruption leads to an increase in epidermal lipid and DNA synthesis. Here we investigate whether barrier disruption also influences keratins and cornified envelope proteins as major structural keratinocyte proteins. Cutaneous barrier disruption was achieved in hairless mouse skin by treatments with acetone +/- occlusion, sodium dodecyl sulfate, or tape-stripping. As a chronic model for barrier disruption, we used essential fatty acid deficient mice. Epidermal keratins were determined by one- and two-dimensional gel electrophoresis, immunoblots, and anti-keratin antibodies in biopsy samples. In addition, the expression of the cornified envelope proteins loricrin and involucrin after barrier disruption was determined by specific antibodies in human skin. Acute as well as chronic barrier disruption resulted in the induction of the expression of keratins K6, K16, and K17. Occlusion after acute disruption led to a slight reduction of keratin K6 and K16 expression. Expression of basal keratins K5 and K14 was reduced after both methods of barrier disruption. Suprabasal keratin K10 expression was increased after acute barrier disruption and K1 as well as K10 expression was increased after chronic barrier disruption. Loricrin expression in mouse and in human skin was unchanged after barrier disruption. In contrast, involucrin expression, which was restricted to the granular and upper spinous layers in normal human skin, showed an extension to the lower spinous layers 24 h after acetone treatment. In summary, our results document that acute or chronic barrier disruption leads to expression of keratins K6, K16, and K17 and to a premature expression of involucrin. We suggest that the coordinated regulation of lipid, DNA, keratin, and involucrin synthesis is critical for epidermal permeability barrier function.

Topics: Animals; Cell Differentiation; Cell Division; Epidermis; Female; Humans; Keratins; Membrane Proteins; Mice; Mice, Hairless; Permeability; Protein Precursors; Time Factors

Basal keratins, suprabasal keratins, filaggrin, and cornified cell envelope (CCE) precursor proteins are expressed during the differentiation of epidermal keratinocytes. These molecules are coordinately expressed during epidermal differentiation. The present study investigated the expression patterns of keratins and CCE precursor proteins in 15 patients with epidermolysis bullosa simplex (EBS), which is caused by mutations in the genes that encode for the basal keratins, keratins 5 and 14. The patterns of expression of keratins 5, 14, 1 and 10, filaggrin, and of the three major CCE precursor proteins, involucrin, loricrin and small proline-rich proteins 1 and 2 (SPRs), were studied immunohistochemically and by electron microscopy. In 14 of the 15 patients with EBS, the distribution pattern of keratins was not altered. In one neonate with EBS, basal cell keratins were expressed in the suprabasal layers. Ultrastructurally, numerous clumped tonofilaments were observed in the basal and suprabasal cells. In all cases, findings were positive for filaggrin in the granular cells, with positivity for involucrin in the upper spinous and granular cells. The upper spinous cells and granular cells were positive for SPRs 1 and 2, and loricrin was expressed in granular cells. Ultrastructurally, no marked abnormality was observed in the suprabasal layers such as a decrease in, or agglutination of, keratin filaments, except in one neonate. A CCE about 15 nm thick was formed normally in the cell membrane of cornified cells. The patterns of distributions of basal cell keratins, suprabasal keratins, filaggrin, and CCE precursor proteins, as well as the ultrastructural findings, resembled those of normal skin. Thus, the abnormality in basal cell keratins in patients with EBS did not appear to alter the patterns of expression of the keratins and CCE precursor proteins.

Topics: Adolescent; Adult; Child; Child, Preschool; Epidermis; Epidermolysis Bullosa Simplex; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Infant; Intermediate Filament Proteins; Keratinocytes; Keratins; Male; Membrane Proteins; Middle Aged; Protein Precursors

To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features.. Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out.. 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas.. HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma.

Topics: Aged; Aged, 80 and over; Base Sequence; Blotting, Southern; Carcinoma, Adenosquamous; DNA, Viral; Humans; In Situ Hybridization; Keratins; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Papillomaviridae; Polymerase Chain Reaction; Protein Precursors

The biologically active vitamin D analog calcipotriol is effective and safe in the topical treatment of psoriasis, but its exact mechanism of action is unknown.. We investigated expression of 1,25-dihydroxyvitamin D3 receptors, markers for inflammation (CD1a, CD4, CD8, CD11b, CD15; NAP-1/interleukin-8; 55 kd tumor necrosis factor-receptor; intercellular adhesion molecule-1; HLA-DR), proliferation (proliferating cell nuclear antigen, Ki-67), and differentiation (transglutaminase K; involucrin; cytokeratin 16) in psoriatic skin during topical calcipotriol treatment.. For immunohistochemical staining we used the labeled avidin-biotin technique on cryostat-cut sections.. We found a significant increase of 1,25-dihydroxyvitamin D3 receptor expression in epidermal basal keratinocytes of lesional psoriatic skin during calcipotriol treatment. In all patients analyzed, effects on proliferation and differentiation of epidermal keratinocytes were stronger than effects on dermal inflammation. Effects on inflammation were more pronounced in the epidermal than in the dermal compartment.. Our findings indicate that analogs of 1,25-dihydroxyvitamin D3 upregulate their corresponding receptor in human keratinocytes in vivo. This mechanism may be important in the therapeutic efficacy of vitamin D analogs in psoriasis. The differential therapeutic effects in the epidermal and dermal skin compartments may be due to a reduced bioavailability of calcipotriol in the dermal compartment.

Topics: Administration, Cutaneous; Antigens, CD; Antigens, CD1; Biological Availability; Calcitriol; CD11 Antigens; CD4 Antigens; CD8 Antigens; Cell Differentiation; Cell Division; Dermatologic Agents; Epidermis; Gene Expression Regulation; HLA-DR Antigens; Humans; Immunoenzyme Techniques; Immunohistochemistry; Intercellular Adhesion Molecule-1; Interleukin-8; Keratinocytes; Keratins; Lewis X Antigen; Male; Proliferating Cell Nuclear Antigen; Protein Precursors; Psoriasis; Receptors, Calcitriol; Receptors, Interleukin; Receptors, Interleukin-8A; Receptors, Tumor Necrosis Factor; Skin; Transglutaminases; Tumor Necrosis Factor-alpha; Up-Regulation

To document the cytokeratin expression patterns in the normal human conjunctival epithelium obtained directly from patients using impression cytology.. Impression cytology specimens were obtained from normal volunteers using pure nitrocellulose membranes rather than cellulose acetate. The 31 volunteers of both sexes ranged in age from 18 to 79 years. Impression cytology specimens were analyzed for individual cytokeratins by either immunocytochemistry or electrophoresis with immunoblotting using a defined panel of monoclonal antibodies.. Using the corroborative methods of immunocytochemistry and electrophoresis with immunoblotting, cytokeratins characteristic of nonkeratinized, stratified (K4 and K13), simple (K8 and K19), and glandular epithelia (K7) were present in the superficial layer (s) of normal human conjunctiva. Cytokeratins typical of keratinized epithelia (K1, K2, and K10) and the keratinization-related proteins filaggrin and involucrin were not expressed in normal conjunctival epithelium.. The normal human conjunctiva demonstrates a unique cytokeratin expression pattern containing cytokeratins characteristic of nonkeratinized, stratified epithelia, as well as others more typical of a simple differentiation pattern, a glandular differentiation pattern, or both. These findings provide a foundation for examining changes in the cytokeratin expression pattern in diseased human conjunctival epithelium using impression cytology.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Biomarkers; Blotting, Western; Conjunctiva; Cytological Techniques; Electrophoresis, Polyacrylamide Gel; Epithelium; Female; Filaggrin Proteins; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Protein Precursors

The peripheral changes in the uninvolved skin adjacent to the extending psoriatic lesions may represent early events. The sequence of these events remains controversial. In the present study we evaluated epidermal and dermal aspects of the margin of the progressive psoriatic plaque, the distant uninvolved skin and normal healthy skin, using immunohistochemistry with markers for keratinization, proliferation and connective tissue: filaggrin, involucrin, Ki-67 and tenascin. The results showed that: (i) processes in distant uninvolved skin were comparable with the observations in normal skin; (ii) in the margin zone of the spreading psoriatic lesion, following the increased appearance of tenascin, the transition into parakeratosis, abnormal expression of filaggrin, involucrin and recruitment of cycling epidermal cells, happened simultaneously. The simultaneous normalization of these epidermal processes might be a consequence of a signal which is simultaneously transduced to the basal and suprabasal cell layers of the epidermis. Based on the present results and earlier findings, we would like to propose a triple stage model for the development of the psoriatic lesion: Stage 1, involvement of the stroma; stage II, inflammatory infiltrate formation and penetration into the upper layers of the epidermis, with suprabasal expression of keratin 16; stage III, recruitment of cycling epidermal cells and abnormal terminal differentiation. Further studies are required on the regulation of tenascin expression, focusing on factors derived from the stroma affecting both recruitment of cycling epidermal cells, involucrin and filaggrin expression. An intermediate step in the dermo-epithelial interrelation is the inflammatory infiltrate, penetrating into the most superficial zone of the epidermis, and the suprabasal expression of keratin 16.

Topics: Adult; Aged; Biopsy; Cell Division; Epidermal Cells; Epidermis; Female; Filaggrin Proteins; Gene Expression Regulation; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Ki-67 Antigen; Male; Middle Aged; Protein Precursors; Psoriasis; Skin; Tenascin

The potent calciotropic hormone calcitriol (1,25-dihydroxyvitamin D3, 1,25(OH)2D3) has been shown to be very effective and safe in the topical treatment of psoriasis. In vitro, 1,25(OH)2D3 inhibits proliferation and stimulates differentiation of human keratinocytes. Increasing evidence suggests an immunoregulatory function of this potent steroid hormone. To further characterize the biological effects of topical calcitriol treatment in psoriasis, we have analyzed immunohistochemically the expression of markers for epidermal proliferation (proliferating cell nuclear antigen=PCNA) and differentiation (transglutaminase K, involucrin, cytokeratin 16), as well as inflammation (CD1a, 55 kDa TNF-receptor, NAP-1/IL-8) in calcitriol-treated psoriatic skin in situ. Our findings strongly support the hypothesis that calcitriol modulates keratinocyte proliferation/differentiation as well as inflammation in human skin in vivo. The immunoreactivity of markers for epidermal proliferation and differentiation, as well as of CD1a and NAP-1/IL-8, changed after 8 weeks of calcitriol treatment almost completely to the pattern characteristic for non-lesional psoriatic skin, while a large number of 55 kDa TNF-receptor positive cells could be found in the dermal compartment.

Topics: Administration, Topical; Antigens, CD1; Calcitriol; Cell Differentiation; Cell Division; Humans; Immunohistochemistry; Interleukin-8; Keratinocytes; Keratins; Proliferating Cell Nuclear Antigen; Protein Precursors; Psoriasis; Receptors, Tumor Necrosis Factor; Skin; Transglutaminases

The expression of cytokeratins (CK), involucrin, vimentin, CD34, and alpha-smooth-muscle actin was studied in fetal and adult hair follicles. The first stage of the developing hair follicle is characterized by palisaded, elongated epithelial cells budding from the epidermal basal layer. These cells express CK5/6, CK14, CK17, CK19, and vimentin. During the following weeks of gestation, different structures in the developing hair follicle can be identified and characterized. The matrical cells display only CK19. The keratinocytes of the outer root sheath express CK5/6, CK14, CK17, CK19, and involucrin; those of the inner root sheath, CK4, CK18, and involucrin; those of the isthmus, the same profile as the ORS. In the infundibulum, the basal-layer keratinocytes express CK5/6, CK14, CK17, and CK19, whereas in the suprabasal layers CK1, CK4, CK10, CK14, and CK17 are seen. The adult hair follicle in anagen fails to express CK19 in the matrical cells and isthmus and both CK17 and CK19 in the infundibulum. These profiles of intermediate filaments and other markers appear to be potentially useful in categorizing neoplasms with apparent follicular differentiation.

Topics: Actins; Adult; Antibodies; Antigens, CD34; Epidermis; Epithelium; Fetus; Fixatives; Formaldehyde; Gene Expression Regulation; Gene Expression Regulation, Developmental; Hair Follicle; Humans; Immunohistochemistry; Intermediate Filaments; Keratinocytes; Keratins; Paraffin Embedding; Protein Precursors; Tissue Fixation; Vimentin

Trichoblastoma and nodular basal cell carcinoma are generally held to be distinctive epithelial neoplasms with some overlapping features. We investigated 30 trichoblastomas in which the basaloid cells expressed cytokeratins (CK) CK5/6, CK14, CK17, CK19, and, in a few cells, vimentin. The cells of the periphery of small and large cysts showed the same profile. Cells lining the lumen of small cysts expressed CK14, CK17, and involucrin, and those in larger cysts showed a positivity for CK1, CK4, CK10, CK14, CK17, and involucrin. The remaining tested antibodies (CK7, CK8, CK13, CK18, CK20, alpha-smooth-muscle actin) were negative in all cases. The cells of the stroma expressed vimentin and in 22 cases, the CD34 antigen. Seventeen nodular basal cell carcinomas showed exactly the same staining pattern. Furthermore, there are striking immunohistochemical similarities between the neoplastic basaloid cells of both neoplasms and the cells of the hair germ. Therefore, trichoblastoma and nodular basal cell carcinoma cannot be distinguished by their pattern of cytokeratin expression in paraffin sections. The virtually identical cytokeratin pattern seen in trichoblastoma, basal cell carcinoma, and the developing fetal hair follicle is compelling evidence for common differentiation pathway.

Topics: Actins; Adult; Aged; Aged, 80 and over; Antigens, CD34; Carcinoma, Basal Cell; Cell Differentiation; Cysts; Epithelium; Female; Fetus; Gene Expression Regulation, Neoplastic; Hair Follicle; Humans; Immunohistochemistry; Intermediate Filaments; Keratins; Male; Melanocytes; Middle Aged; Neoplasms, Basal Cell; Paraffin Embedding; Protein Precursors; Skin Neoplasms; Vimentin

We found a carcinoembryonic antigen (CEA)-related antigen to be strongly expressed on a subset of follicular keratinocytes in normal human skin. The antigen was characterized immunohistochemically using a panel of antibodies against human CEA and CEA-related molecules. The expression of the antigen was studied in different phases of the hair cycle as well as in different hair types. Immunohistochemically, the antigen resembled the nonspecific crossreacting antigen (NCA) NCA-50/90 rather than true CEA. Its expression was limited to the innermost cells of the lowest segment of hair follicles in the catagen/telogen phases, being detected only where the hair shaft was attached to the epithelial hair sac in these phases. The same results were obtained for all hair types, i.e. terminal, vellus and intermediate hair. Coexpression of the antigen with both involucrin and differentiation-associated cytokeratins was noted in the cells in additional studies attempting to identify the exact subpopulations of follicular keratinocytes expressing the antigen in comparison with the expression of other functional markers. However, involucrin and the cytokeratins were also expressed in the upper segments of anagen as well as catagen/telogen hair follicles. Our findings strongly suggest that an NCA-50/90-like molecule is expressed cyclically on the innermost cells in the lowest segment of the outer root sheath only in catagen/telogen hair follicles. The cyclical expression in this specific subset of follicular keratinocytes only, in which the epithelial hair sac is attached to the hair shaft, may be associated with the stability of the attachment through the adhesive or, conversely, the repulsive function of CEA-related molecules, both of which have recently been proposed.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Carcinoembryonic Antigen; Cell Adhesion Molecules; Hair Follicle; Humans; Keratinocytes; Keratins; Membrane Glycoproteins; Mice; Proliferating Cell Nuclear Antigen; Protein Precursors

The hallmarks of dry skin (xerosis) are scaliness and loss of elasticity. Decreased hydration and a disturbed lipid content of the stratum corneum are also well-known features. The frequency of dry skin increases with ageing. The aim of this study was to examine if these known features of dry skin are related to changes in epidermal proliferation and differentiation. In addition, age-related changes in normal and in dry skin were examined: 62 volunteers were divided by clinical grading and biophysical measurements into groups with young/normal, young/dry, aged/normal and aged/dry skin. Biopsy samples from the lower legs (most severe dryness) were examined by two-dimensional gel electrophoresis and by immunohistochemistry for epidermal proliferation, epidermal keratins and cornified envelope proteins. There was a slight increase in proliferation in both groups with dry skin compared with normal skin of the corresponding age. In aged/normal compared with young/normal skin there was a significant decrease in proliferation. However, epidermal proliferation was the same in aged/dry skin as in young/normal skin. For epidermal differentiation, an age-independent decrease of keratins K1 and K10 and an associated increase in the basal keratins K5 and K14 was detected in dry skin. There was also an age-independent premature expression of the cornified envelope protein involucrin. In contrast, loricrin expression was not influenced by dry skin conditions. In summary, epidermal proliferation was significantly decreased in aged/normal compared with young/normal skin. Dry skin showed significant changes in the epidermal expression of basal and differentiation-related keratins, and a premature expression of involucrin irrespective of age.

Topics: Adult; Aged; Aging; Body Water; Cell Differentiation; Cell Division; Electrophoresis, Gel, Two-Dimensional; Epidermis; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Protein Precursors; Skin Diseases; Water Loss, Insensible

We have established and characterized three cell lines from normal human vaginal, ectocervical, and endocervical epithelia immortalized by expression of human papillomavirus 16/E6E7. The lines (VK2/E6E7, Ect1/E6E7, and End1/E6E7) displayed distinctive morphologies at the level of light microscopy when cultured in calcium-supplemented (0.4 mM) keratinocyte serum-free medium and maintained a stable phenotype after more than 1 yr of continuous passage. They were compared to primary cell cultures and epithelial cells in sections of the respective native tissues for expression of epithelial differentiation proteins. All cell lines expressed cytokeratin (CK) 8, CK18, and CK19, and some cells in all three cell lines expressed CK16, involucrin, and the secretory component of the polymeric immunoglobulin receptor. The vaginal and ectocervical cell lines expressed CK10 and CK13, whereas the endocervical line did not. With the exception of CK8 and CK18 expression, the morphological and immunocytochemical characteristics of the immortalized lines closely resembled those of their respective tissues of origin and primary cultures, and all differed significantly from the HeLa cervical adenocarcinoma cell line. These new cell lines may provide the basis for valid, reproducible in vitro models for studies on cervicovaginal physiology and infections and for testing pharmacological agents for intravaginal application.

Topics: Antigens, Differentiation; Cell Line; Cervix Uteri; Epithelial Cells; Female; Humans; Immunohistochemistry; Keratins; Papillomaviridae; Phenotype; Protein Precursors; Receptors, IgG; Vagina

Oral keratinocytes originate from basal cells, differentiate during migration to the surface, and finally are shed. Apoptosis occurs at the end of differentiation, but the precise relationship between terminal differentiation and apoptosis is not clear. In the present study, Bcl-xL was expressed in the basal cell and spinous cell layers, and Bax was expressed in the spinous cell and granular cell layers. In cultured keratinocytes, Bcl-xL was expressed under conditions of 0.1 mM calcium (low Ca2+) but disappeared under conditions of 1.0 mM calcium (high Ca2+); the latter induces keratinocyte differentiation. Bax was not expressed in keratinocytes with low Ca2+ but was expressed in cells with high Ca2+. Finally keratinocytes with high Ca2+ underwent apoptosis, which was detected by the TUNEL method and by 180-bp DNA fragmentation. These results suggest that the process of terminal differentiation in gingival epithelium is a pathway to apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Cell Differentiation; Cells, Cultured; Culture Media; DNA Nucleotidylexotransferase; Electrophoresis, Agar Gel; Epithelial Cells; Epithelium; Gingiva; Humans; Keratinocytes; Keratins; Protein Precursors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

To investigate the histogenesis of mixed tumor of the skin, apocrine type, the immunophenotypes of 10 cases were examined using 19 different monoclonal anti-keratin antibodies and antibodies against carcinoembryonic antigen (CEA) and involucrin. By using light microscopy, four epithelial elements in this tumor were characterized: tubular branching structures with lumina lined by cuboidal epithelium and those with lumina lined by columnar epithelium, keratinous cysts, and solid aggregates of epithelial cells. The immunohistochemical patterns of cytokeratin expression suggested that cuboidal and columnar cells differentiated, respectively, toward the ductal and secretory cells of apocrine glands, whereas keratinous cysts revealed follicular infundibular differentiation. Furthermore, CEA expression, a marker for sweat-gland differentiation, was present not only on tubules' luminal surfaces but also on the inner surfaces of keratinous cysts. The simultaneous coexpression of CEA and cytokeratins specific for follicular infundibulum in the keratinous cysts, although perplexing, suggested that keratinous cysts may contain some cells differentiating toward the intrafollicular portion of apocrine ducts that enter infundibulae rather than eccrine ducts that have no infundibular association. We conclude that apocrine type of mixed tumors of the skin demonstrate differentiation toward all components of apocrine units.

Topics: Adenoma, Pleomorphic; Apocrine Glands; Carcinoembryonic Antigen; Cell Differentiation; Cell Lineage; Cysts; Eccrine Glands; Epithelial Cells; Epithelium; Gene Expression Regulation, Neoplastic; Hair Follicle; Humans; Immunohistochemistry; Immunophenotyping; Keratins; Middle Aged; Protein Precursors; Skin Neoplasms; Sweat Glands

In the present study, keratin and involucrin expression were studied in cutaneous lesions of discoid lupus erythematosus and lichen planus in order to gain a better understanding of the abnormal differentiation or maturation of the epidermal cells in these dermatoses. Ten specimens each from discoid lupus erythematosus and lichen planus were analyzed by immunohistochemical techniques, using a panel of monoclonal antikeratin antibodies and polyclonal anti-involucrin antibody, and five specimens each were analyzed by one- and two-dimensional gel electrophoresis and immunoblot analysis using three antikeratin antibodies. No significant difference was found between the dermatoses. The expression of differentiation-specific keratins showed a similar pattern to that in normal epidermis, and involucrin was expressed even in the lower part of the stratum spinosum. Keratins 6 and 16, which are characteristic markers of hyperproliferative states, and keratin 17 were detected in nonhyperproliferative and atrophic epidermis with hydropic degeneration and inflammatory infiltrates in the dermis. These results suggest that expression of keratins 6, 16 and 17 in discoid lupus erythematosus and lichen planus may reflect a wound healing response to the damage to the basal cell layer, or may be under the control of cytokines produced by infiltrating inflammatory cells in the dermis.

Topics: Cell Differentiation; Cellular Senescence; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Immunoenzyme Techniques; Keratins; Lichen Planus; Lupus Erythematosus, Discoid; Protein Precursors

The objective of this investigation was to study the relationship of the ghost cell ameloblastoma (GCA), which is a form of type II calcifying odontogenic cyst (COC), to the adamantinomatous craniopharyngioma (ACP). H&E sections of 26 examples of ACP were compared to three cases of GCA and to the reported microscopic features of that tumor. Clinical records of the ACPs were studied to determine their biologic behavior compared to that of the ameloblastomas. Immunohistochemical studies of nine examples of ACP were performed for KL1 (high mol.wt cytokeratins), 5D3 (low mol.wt cytokeratins) and involucrin (characteristic of terminally differentiated keratinocytes) using the peroxidase-antiperoxidase method. The results were compared with those reported for COC and ameloblastoma. ACP and GCA exhibited similar microscopic features, including pre-ameloblasts, tissue resembling stellate reticulum, ghost cells and calcifications; both tumors grew slowly and were invasive. ACP and COC, and by interpolation GCA, exhibited similar features with all three antibodies. The ghost cells did not exhibit any immunoreactivity but the adjacent cells stained positively for involucrin. The immunological features of ACP were similar to those reported in ameloblastomas for squamous differentiation. However, because of their rarity, no ameloblastomas exhibiting keratinization, including ghost cells, have yet been studied with these antibodies. We conclude that ACP and GCA are homologous lesions.

Topics: Ameloblastoma; Ameloblasts; Biology; Calcinosis; Cell Differentiation; Coloring Agents; Craniopharyngioma; Eosine Yellowish-(YS); Epithelial Cells; Fluorescent Dyes; Hematoxylin; Humans; Immunoenzyme Techniques; Immunohistochemistry; Jaw Neoplasms; Keratinocytes; Keratins; Molecular Weight; Odontogenic Cyst, Calcifying; Pituitary Neoplasms; Protein Precursors

Four cases of resected adenosquamous carcinoma of the liver were clinicopathologically reviewed, together with immunohistochemical findings. Although no lymph node metastases were seen and a curative resection was achieved in all cases, two patients had recurrences in the peritoneum and distant organs such as the pericardium and pleura relatively soon after the operation. Of the remaining two cases, one patient died during the postoperative period and the other died of coexistent hilar cholangiocarcinoma. Together these findings suggest that this disease tends to spread locally and distantly in the early phase of tumor growth and shows aggressive biological behavior. In an immunohistochemical study, involucrin was a specific marker for the squamous component and CA19-9 was a marker for the adenomatous component.

Topics: Aged; CA-19-9 Antigen; Carcinoembryonic Antigen; Carcinoma, Adenosquamous; Female; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Male; Protein Precursors; Treatment Outcome

Bronchial epithelial dysplasia is a non-invasive cellular change often associated with physical or chemical injury and considered a pre-neoplastic lesion in the formation of lung cancer. A series of 39 bronchial dysplasias associated with both neoplastic and non-neoplastic lesions were assessed for expression of markers of differentiation by immunocytochemistry and compared with samples of normal bronchial epithelium. The normal bronchial epithelium studied expressed cytokeratins (CKs) 4, 6, 7, 8, 18, and 19 in all cases; CK 13 in 13 cases; and peanut agglutinin (PNA) in seven cases. Involucrin, CK 10, and CK 14 were not observed in the normal bronchial samples. In the dysplastic bronchial biopsies, epithelial staining was observed with epithelial CKs 7, 8, 18, and 19 in all cases; CK 13 was seen in 26 cases; CK 14 in 13 cases; CK 6 in 11 cases; and CK 10 in five cases. In 13 cases of dysplasia, only simple epithelial antigens were identified. Involucrin expression was observed in 17 dysplastic biopsies and PNA in 12. By Fisher's exact test, a significant association between non-severe histological grade of dysplasia and CK 6 expression (P = 0.018) was found. Comparison of the results using the same analysis showed significant correlations between the loss of CK 6 expression (P < 0.001) and the expression of CK 14 (P = 0.008) and involucrin (P = 0.0018) with bronchial dysplasia. These data show that the pattern of differentiation antigen expression in bronchial dysplasia is significantly different from that of the normal bronchial epithelium, but the phenotypic heterogeneity of these lesions is similar to that of bronchial carcinomas.

Topics: Aged; Aged, 80 and over; Antigens, Differentiation; Biomarkers; Bronchi; Cell Differentiation; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Lectins; Lung Neoplasms; Middle Aged; Peanut Agglutinin; Precancerous Conditions; Protein Precursors

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/ elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs.

Topics: Blotting, Northern; Carrier Proteins; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Culture Media; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Flow Cytometry; Humans; Immunohistochemistry; Keratinocytes; Keratins; Membrane Proteins; Myelin P2 Protein; Neoplasm Proteins; Phenotype; Protein Precursors; Proteinase Inhibitory Proteins, Secretory; Proteins; Psoriasis; RNA, Messenger; Serine Proteinase Inhibitors; Transglutaminases; Tumor Suppressor Proteins

Eccrine syringofibroadenoma is a rare benign skin tumor, which usually develops on the extremities of elderly persons. We performed immunohistochemical and ultrastructural studies of a typical case of eccrine syringofibroadenoma that developed on the left heel of a 58-year-old man. The tumor consisted of anastomosing thin epithelial strands connected to the epidermis. There were many ductal or cystic structures, and their luminal cells were strongly positive to antibodies against carcinoembryonic antigen and epithelial membrane antigen. Filagrin and involucrin immunoreactivities were also detected in some cells surrounding the ducts. Keratins K1 and K10, co-expressed in the peripheral cells of normal acrosyringia, were colocalized in small cell clusters. Ultrastructurally, intracellular duct formation characteristic of developing acrosyringia was observed. Tumor cells containing globular keratohyaline granules with various electron densities were seen around some ductal structures. In these areas, keratinization took place without lamellar granule formation or prominent cornified cell envelope assembly. These results suggest acrosyringial differentiation of this tumor.

Topics: Adenoma, Sweat Gland; Antigens, Neoplasm; Carcinoembryonic Antigen; Cytoplasmic Granules; Eccrine Glands; Epidermis; Epithelium; Filaggrin Proteins; Foot Diseases; Humans; Hyalin; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Mucin-1; Protein Precursors; Sweat Gland Neoplasms

A 47-year-old woman noticed a nodule on her right shoulder that had been gradually increasing in size without symptoms. Histologic features of the biopsied nodule included round to irregularly shaped epithelial lobules demarcated by abundant sclerotic stroma located within the lower dermis and extending to the subcutis. The epithelial lobules consisted of cuboidal to columnar basaloid cells and were frequently arranged in narrow strands with many bifurcations and branching. Cystic structures containing lamellar keratinous material were occasionally found in connection with the lobules. The histologic findings were interpreted as trichoblastic fibroma. Immunohistochemical studies with antibodies directed against cytokeratins (CK) and involucrin revealed positive staining in most of the tumor cells with RCK102 and 34 beta E12 antikeratin antibodies, whereas the epithelial cords and the peripheral cells of the cystic structures stained with 170.2.14, 4.1.18, and CAM 5.2 antikeratin antibodies. However, CK1 or simple epithelial cytokeratins were not detected in any neoplastic elements. Based on comparative immunohistochemical findings in normal hair follicles, we propose that trichoblastic fibroma may first differentiate toward the outermost cell layer of the outer root sheath between the lower permanent portion and the upper transient portion and then into various other parts of the hair follicle.

Topics: Epithelium; Female; Fibroma; Gene Expression Regulation, Neoplastic; Hair Follicle; Humans; Immunohistochemistry; Keratins; Middle Aged; Protein Precursors; Sclerosis; Shoulder; Skin; Skin Neoplasms

We examined full thickness specimens of oesophageal squamous dysplasia from both cancer-free and cancer patients using immunohistochemical labelling for cytokeratin subtypes 10/13 and 14 and for involucrin, binding studies for various lectins, and PAS/D staining before and after diastase treatment. We studied specimens from patients with oesophageal carcinoma (52 normal epithelia, and 49 with mild, 38 with moderate, and 32 with severe dysplasia), and 32 specimens from cancer-free patients (five normal epithelia and 16 with mild and 11 with moderate dysplasia). Abnormal cytokeratin expression patterns in atypical cells, i.e. both cytokeratin 10/13 and cytokeratin 14 immunoreactivity in the same cells was detected in 41 of 99 specimens with dysplasias in cancer patients. Helix aspersa, Erythrina cristagalli and Robinia pseudoacacia binding was consistently negative in atypical cells in squamous dysplasia. The non-atypical layer of squamous dysplasia, which was morphologically indistinguishable from the corresponding layer of normal oesophageal squamous epithelium, showed abnormal involucrin expression in 39/ 101 specimens, Helix aspersa binding in 74/106, diastase sensitive PAS staining in 52/110, Erythrina cristaglli binding in 28/107, and Robinia pseudoacacia binding in 16/100. There were no significant differences in the expression of these markers in dysplasia between cancer patients and cancer-free individuals with the exception of increased Robinia pseudoacacia binding in the non-atypical layer in cancer-free patients. The results indicate that abnormal patterns of cytokeratin expression and lectin binding occur not only in atypical cells but also in non-atypical cells in oesophageal squamous dysplasia.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lectins; Male; Middle Aged; Precancerous Conditions; Protein Binding; Protein Precursors

Although the induction of acute irritant dermatitis by detergents has been studied extensively in recent years, our understanding of the cell biological events in the repair phase, and its relevance for the development of chronic irritant dermatitis is limited. Here we studied the reaction pattern of human skin to short-term application of sodium dodecyl sulphate (SDS) in a model that induced a minimal acute inflammatory reaction (absence of polymorphonuclear leucocytes, PMN) and did not have cytopathic effects on the epidermal keratinocytes as determined by histological investigation. All parameters were measured up to 14 days after exposure to SDS. Application of SDS caused disturbances of barrier function as measured by transepidermal water loss and had vascular effects as judged by erythema. Several cell biological markers for epidermal growth and differentiation were examined by immunohistochemistry. A rapid and strong induction of the cornified envelope precursor protein involucrin was seen in the stratum spinosum, with a peak at 24 h. Within 24 h a strong upregulation of epidermal fatty acid binding protein (E-FABP) was noted, with a peak at 7 days after injury. Cellular proliferation in the basal layer was increased fivefold as assessed by nuclear staining for the Ki-67 antigen, showing a peak at 48 h. Surprisingly, no significant induction of cytokeratin 16 and SKALP/elafin expression, two markers associated with epidermal hyper-proliferation and inflammation, was seen. These findings suggest that the cellular changes following exposure to detergent are distinct from those seen in other forms of skin injury. We would speculate that the epidermal response to detergent exposure is primarily directed at restoration of barrier function.

Topics: Adult; Biomarkers; Carrier Proteins; Cell Differentiation; Cell Division; Dermatitis, Irritant; Detergents; Epidermis; Erythema; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Female; Humans; Keratinocytes; Keratins; Ki-67 Antigen; Male; Models, Biological; Myelin P2 Protein; Neoplasm Proteins; Protein Precursors; Proteinase Inhibitory Proteins, Secretory; Proteins; Sodium Dodecyl Sulfate; Time Factors; Tumor Suppressor Proteins

The detection of cytokeratins in neoplastic tissues by immunohistochemical methods has numerous diagnostic and investigative applications, because cytokeratins are usually conserved in tumour cells during malignant transformation. Recently, however, it has been reported that progression to malignancy is associated with commencement of expression of low-molecular-weight cytokeratins. In the present study, 42 specimens from 35 cases of squamous cell carcinoma (SCC) of the skin were analysed by immunohistochemical techniques, using polyclonal anti-involucrin antibody and a panel of monoclonal antikeratin antibodies, in order to investigate the nature and differentiation of SCCs. The expression of cytokeratins and involucrin in well-differentiated SCCs was similar to that in normal epidermis. In contrast with well-differentiated SCCs, the expression of differentiation-specific cytokeratins and involucrin was diminished in the immature tumour cells in proportion to the malignancy of the SCCs. Some antibodies, however, stained all tumour cells, irrespective of the degree of malignancy. Furthermore, expression of simple epithelial and non-cornifying stratified squamous epithelial cytokeratins was observed in atypical tumour cells of poorly differentiated SCCs. It is of interest that similar expression was noted in many tumour cells in the lymph node metastases and in some tumour cells in the primary cutaneous lesions. Cytokeratin expression similar to that in normal epidermal keratinocytes was conserved in well-differentiated SCCs, but the expression of cytokeratins changed during progression to malignant transformation. The expression of simple epithelial or non-cornifying stratified squamous epithelial cytokeratins in cutaneous SCCs may be a marker for their capability of invasion and metastatic potential.

Topics: Antibody Specificity; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Protein Precursors; Skin Neoplasms

A new patient with CHILD syndrome (congenital hemidysplasia, ichthyosiform erythroderma, and limb defects), the thirtieth in the literature, was observed for over three years. Initially, the right-sided lesion spared the breast area. At 10 months of age the trunk lesion extended to cover the entire area of the right chest. At age 20 months the patient developed linear, bandlike, keratotic, brown-black lesions on her left thigh that subsided within six weeks, leaving a slight hyperpigmentation. This patient was studied by routine histologic methods as well as with markers of keratinization and electron microscopy. In hematoxylin and eosinstained sections, parakeratosis and orthokeratosis alternated. In some parakeratotic areas, large granular cells, and in others, ghost granular cells, were present. The latter showed basophilic cytoplasm, and palestaining or vacuolated nucleus and were seen either above the normal granular layer or without it. Although regional variations existed, basal cell-type keratins as recognized by AE1 continued to be expressed in suprabasal layers. Filaggrin- and involucrin-positive layers were expanded, particularly the latter, down to the lower prickle cell layer. Ultrastructurally, numerous lamellar or membranous structures were found in upper layers of the epidermis, both intracellulary and intercellularly. Normal cementsomes coexisted with these abnormal lamellar structures, and it was thought that the latter represent modified cementsomes because the discharge of those from the cell periphery was often detected.

Topics: Arm; Epidermis; Female; Filaggrin Proteins; Follow-Up Studies; Humans; Hyperpigmentation; Ichthyosiform Erythroderma, Congenital; Infant; Infant, Newborn; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratosis; Leg; Lichenoid Eruptions; Protein Precursors; Syndrome

The nature of clear cell acanthoma has not been clarified, although many hypotheses have been proposed, including a benign neoplasm derived from epidermis or the acrosyringium, or a non-specific dermatosis. In this study, seven cases of clear cell acanthoma were analysed by immunohistochemical techniques, using various monoclonal antikeratin antibodies, and antibodies against filaggrin, involucrin and epithelial membrane antigen. Different immunoreactivities were noted between clear cell acanthoma and a normal eccrine gland, including the acrosyringium. Immunoreactivities of clear cell acanthoma were almost identical to those of normal epidermis, although some antibodies gave a different staining pattern between clear cell acanthoma and normal epidermis. The expression of cytokeratins in psoriatic epidermis has been reported to change as a result of abnormal differentiation or maturation. Clear cell acanthoma showed a similar staining pattern to inflammatory dermatoses such as psoriasis vulgaris, lichen planus and discoid lupus erythematosus. We speculate that clear cell acanthoma is a localized form of inflammatory dermatosis rather than a neoplasm.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Epidermis; Filaggrin Proteins; Humans; Hyperplasia; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mucin-1; Mucins; Protein Precursors

Allergic and irritant contact dermatitis are similar clinically, histologically and on immunohistochemistry. In the present investigation, we assessed whether study of the recruitment of cycling epidermal cells, and the expression of keratin 16 and involucrin, are of use in differentiating between the response to contact allergens and the response to the irritant detergent sodium lauryl sulphate. Both allergic and irritant challenges induced epidermal proliferation, and the expression of keratin 16 and involucrin, but the dynamics were different. Two and 3 days after challenge, a highly significant difference between the allergic and irritant reactions was observed with respect to involucrin expression assessed by MON-150 staining.

Topics: Adult; Aged; Cell Differentiation; Cell Division; Dermatitis, Allergic Contact; Dermatitis, Irritant; Epidermis; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Protein Precursors; Time Factors

To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation.. Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components.. The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds.. Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding.

Topics: Basement Membrane; Cell Differentiation; Cells, Cultured; Dermatologic Surgical Procedures; Female; Humans; Keratinocytes; Keratins; Male; Mucins; Postoperative Period; Protein Precursors; Skin; Skin Transplantation; Tattooing; Wound Healing

The present study was conducted to determine the patterns of immunohistochemical characterization of keratin (K) and involucrin in solar keratosis and Bowen's disease in order to clarify the abnormal differentiation or maturation of the tumor cells in these precancerous epithelial dermatoses. Seventeen human anti-cytokeratin antibodies and an anti-involucrin antibody were used to examine 15 cases of solar keratosis and 18 cases of Bowen's disease. Formalin-fixed and paraffin-embedded sections were stained with these antibodies by the avidin-biotin-peroxidase technique. In solar keratosis, keratin and involucrin distribution was similar to that in normal epidermis, whereas in Bowen's disease the keratin distribution varied among individual cases. The dyskeratotic cells in Bowen's disease showed a reduction or loss of staining with these antibodies, and they were occasionally positive for keratin 19. These observations suggest that there is a difference in keratin and involucrin expression between solar keratosis and Bowen's disease and that the atypical cells of Bowen's disease exhibit a diversity of differentiation.

Topics: Aged; Aged, 80 and over; Bowen's Disease; Cell Differentiation; Cellular Senescence; Epidermis; Female; Fixatives; Formaldehyde; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunohistochemistry; Keratins; Keratosis; Male; Middle Aged; Paraffin Embedding; Precancerous Conditions; Protein Precursors; Skin Neoplasms; Sunlight

Histologic grade seems to be of limited prognostic significance in patients with vulvar carcinoma. However, the study of cytokeratin expression is of potential interest because it allows a more precise evaluation of the degree of squamous differentiation. This study was conducted to investigate whether differences in cytokeratin expression exist between normal vulvar epithelium and vulvar carcinoma and whether these differences are prognostically significant.. The expression of several differentiation markers, i.e., cytokeratin (CK) 10, CK 13, and involucrin, was studied in samples of 41 vulvar carcinomas. The expression of CK 8, 10, 13, and 14 was compared with CK expression in normal vulvar epithelium and was correlated with tumor grade and tumor growth pattern. Tumor growth pattern was considered type A if infiltrating tumor cell nests showed a layer of small, basaloid cells bordering the surrounding mesenchymal tissue and was considered type B if this was not the case. Prognosis was based on whether disease recurred or not.. Sixteen patients had disease recurrence. No prognostic significance of tumor grade was found. Tumor growth pattern was prognostically significant: in patients with a type A tumor, recurrence was observed less often than in patients with a type B tumor (P = 0.03). Cytokeratin 14, typical for basal cells of normal vulvar epithelium, was expressed in all tumors, whereas CK 8 was not expressed in any tumor. A relationship between tumor growth pattern and the concordant expression of differentiation markers was observed: in 55% of type A tumors and in none of type B tumors, concordant expression of CK 10, CK 13, and involucrin was found.. The expression of differentiation markers in vulvar carcinoma is related strongly to the tumor growth pattern, and this pattern is prognostically significant.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Female; Humans; Immunoenzyme Techniques; Keratins; Multivariate Analysis; Prognosis; Protein Precursors; Recurrence; Vulvar Neoplasms

Normal keratinocytes from epidermis and from buccal mucosa underwent dissimilar stages of differentiation in the same culture medium and responded differently to changes in the composition of the medium. Manifestations of these variations were examined in terms of the expression at the mRNA level (as measured by reverse transcriptase-polymerase chain reaction) of three regulatory genes (cdc2, c-myc, and p53) and five that encode structural proteins (keratins K5, K10 and K13, involucrin, and filaggrin), in three growth-medium formulations. The culture conditions enhanced or retarded maturation; the observed alterations in gene expression correlated with these changes. Except for the proliferation genes, the non-keratinizing buccal mucosa generally responded more weakly than the orthokeratotic epidermis to culture-medium supplementation favouring differentiation. Gene expression in cultured keratinocytes reflected their ability to differentiate in vivo; genes were expressed even when the corresponding protein was not seen in vitro. Although keratin K10 is not prevalent in the buccal mucosa nor keratin K13 in the epidermis, the genes for both were found to be expressed in both tissues.

Topics: Calcium; Cell Differentiation; Cell Division; Cells, Cultured; Culture Media; Culture Media, Serum-Free; Epidermal Cells; Epidermis; Filaggrin Proteins; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Genes, cdc; Genes, myc; Genes, p53; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Mouth Mucosa; Polymerase Chain Reaction; Protein Precursors; RNA, Messenger; Transcription, Genetic

Human papillomaviruses (HPVs) are important human pathogens associated with a range of epithelial neoplasia. The rising incidence of HPV infection and association of HPV with malignancy has led to increased interest in appropriate management of these infections. Development of new therapies for viral warts has been frustrated by the lack of availability of models permissive for viral replication. Here we describe the development of HPV-severe combined immunodeficient mouse model which reproduces mature HPV-infected epithelia. Grafting of anogenital and laryngeal papillomas harbouring either HPV-6 or HPV-11 resulted in the formation of a differentiated neo-epithelium exhibiting the hallmark features of HPV infection including basal hyperplasia, acanthosis and koilocytosis. The reformed warty epithelium contained amplified HPV DNA and expressed capsid protein in the differentiated layers. A striking feature is the production of macroscopic papillomata in an anatomically relevant and accessible site, providing a system of particular relevance for the temporal evaluation of developing lesions and selection of antiviral agents.

Topics: Animals; Capsid; Condylomata Acuminata; Disease Models, Animal; DNA Replication; Epithelium; Gene Expression; Humans; Keratins; Laryngeal Neoplasms; Mice; Mice, SCID; Neoplasm Transplantation; Papilloma; Papillomaviridae; Papillomavirus Infections; Proliferating Cell Nuclear Antigen; Protein Precursors; Skin; Skin Transplantation; Transplantation, Heterologous; Tumor Virus Infections; Virus Replication

Sunburn cells (SBCs) appear in the epidermis shortly after acute UV damage, especially after exposure to UVB light. As yet, the mode of their formation remains to be satisfactorily elucidated. In order to characterize these cells, the expression of various markers of epidermal differentiation following UV exposure was investigated using immunhistochemical procedures. These were applied to paraffin-embedded (microwave technique) and frozen specimens of human skin 24 h after irradiation with 4 times the minimal erythema doses(MED). Normal nonirradiated skin without irradiation served as the control. We used a battery of antibodies directed against the following: cytokeratins (CKs) 5, 10, 17, and 19, actin, cell-adhesion proteins (desmoplakins, desmogleins), markers of terminal epidermal differentiation (filaggrin, involucrin and loricrin), markers of proliferation (PCNA, MIB, K6,16), a marker of endocytosis (clathrin) and markers of cell growth, (transforming growth factor [TGF-alpha]) and B-cell leukemia/lymphoma-2 [bcl-2]. After UV irradiation it was found that CK 5, which is typically confined to basal keratinocytes, was also expressed in suprabasal keratinocytes. The CKs 1 and 10/11 exhibit a normal suprabasal localization, but suprisingly, SBCs were negative for these CKs. Although CK 6,16, and 17 are not usually found in normal epidermis, UVB exposure induced their expression in suprabasal keratinocytes, but again failed to elicit their expression in SBCs. Antibodies specific for markers of late epidermal differentiation (filaggrin, involucrin and loricrin), cell-junction proteins (desmogleins, desmoplakins), proliferation (PCNA and MIB), and endocytosis (clathrin) also failed to produce positive staining of SBCs. Even though TGF-alpha immunoreactivity became detectable in most keratinocytes after UV exposure, this was not the case for SBCs. The number of basally located dendritic cells, most probably melanocytes, exhibiting bcl-2 staining was markedly reduced 6 and 12 h after irradiation as compared with normal skin. SBCs do not express any late differentiation markers, but they do contain proteins typical of basal keratinocytes (CK 5). It can be concluded that SBCs do not develop beyond a more basal-like differentiation pattern, probably as a result of cell death and migration through the epidermis.

Topics: Actins; Antigens, Nuclear; Biomarkers; Cell Adhesion Molecules; Cell Differentiation; Cell Division; Clathrin; Cytoskeletal Proteins; Dendritic Cells; Desmogleins; Desmoplakins; Endocytosis; Epidermis; Filaggrin Proteins; Gene Expression Regulation; GTP-Binding Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Melanocytes; Membrane Proteins; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Protein Precursors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Sunburn; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Retinoids have been shown to modulate the expression of proteins involved in epidermal differentiation. To examine this effect in an in vitro skin model, we evaluated the effect of retinoic acid on the expression of two cell envelope proteins, loricrin and involucrin, and an early marker of epidermal differentiation, keratin 1, in a reconstituted human skin equivalent cultured at the liquid-air interface. Retinoic acid, a known inhibitor of keratinization in monolayer and raft cultures, was evaluated for its ability to alter the expression and distribution of these markers of epidermal differentiation. Retinoic acid (10(-6) M) suppressed loricrin expression in skin cultures as determined by both protein and mRNA analysis. In contrast, no inhibition of involucrin or K1 expression was observed at the protein level at the same retinoic acid concentration. However, some suppression of K1 mRNA transcription was observed in retinoic acid-treated cultures. These results demonstrate that in differentiating cultures of reconstituted human skin, loricrin expression is markedly inhibited by retinoids, K1 less so, and involucrin not at all.

Topics: Base Sequence; Cells, Cultured; Gene Expression; Humans; Immunohistochemistry; Keratins; Membrane Proteins; Molecular Sequence Data; Protein Precursors; RNA, Messenger; Skin; Skin Physiological Phenomena; Tretinoin

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS.cm-2, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenoty

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Transformed; Cell Polarity; Cells, Cultured; Ceramics; Cervix Uteri; Collagen; Culture Techniques; Epithelial Cells; Epithelium; Female; Humans; Hydrocortisone; Keratins; Permeability; Polytetrafluoroethylene; Protein Precursors; Tretinoin; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vimentin

Topics: Adult; Aged; Antibodies, Monoclonal; Conjunctiva; Cytological Techniques; Epithelium; Eye Proteins; Female; Filaggrin Proteins; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Protein Precursors

The identification of pretreatment markers with predictive significance for the presence of lymph node metastases and treatment outcome in low stage cancer of the uterine cervix is clinically important. Because the presence of differentiation-related markers varies in this type of cancer, the authors investigated whether loss of these markers is related to a poor clinical course.. An indirect immunoperoxidase technique was applied to formalin fixed, paraffin embedded tissue sections of 80 patients with International Federation of Gynecology and Obstetrics Stage IB and IIA primary squamous cell cervical carcinomas for detection of expression of cytokeratin 10 and 13, and involucrin. Comparisons were made of the expression of each of these markers among 40 patients with regional node metastases and 40 age-matched patients with no lymph node metastases. Differences in the frequency of expression of these markers also were analyzed in relation to histopathologic characteristics, recurrence, and survival.. Expression of cytokeratin 10, 13, and involucrin was found in 24, 64, and 53%, respectively, of all patients studied. The authors found no differences between patients with positive regional lymph nodes and those with negative lymph nodes. Expression of cytokeratin 13 and involucrin was associated with tumor grade (P = 0.01). No relationship was found between expression of the markers used and recurrence or survival in the entire group. Within the lymph node-positive group, however, the survival rate of patients with tumors with cytokeratin 13 expression was significantly higher than that of patients with tumors lacking cytokeratin 13 expression (P = 0.02).. Expression of cytokeratin 10, 13, or involucrin in the primary tumor is of no predictive value with respect to the presence of regional lymph node metastases in low stage squamous cell cervical cancer. However, cytokeratin 13 expression appears to be of prognostic significance in patients with positive regional lymph nodes.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Protein Precursors; Retrospective Studies; Uterine Cervical Neoplasms

The ability of all-trans-retinoic acid (RA) to modulate the growth and squamous differentiation of four head and neck squamous cell carcinoma cell lines (183, 886, 1483, and SqCC/Y1) was examined, and the relationship of their state of squamous differentiation and RA responsiveness to the expression of cytosolic RA-binding proteins (CRABPs), nuclear RA receptors (RARs), and retinoid X receptors (RXRs) was investigated. RA inhibited proliferation of all but the 183 cell line and suppressed squamous differentiation markers K1 keratin, type 1 transglutaminase, and involucrin mRNAs and proteins to varying degrees in 183, 1483, and SqCC/Y1 cells. Traces of CRABP-I mRNA were detected only in the 886 cells, whereas CRABP-II mRNA was detected in the other three cell lines. RA suppressed CRABP-II expression in SqCC/Y1 cells but had no effect on its expression in the other cell lines. All cell lines expressed mRNAs for RAR-alpha, RAR-beta, RAR-gamma, and RXR-alpha. The RAR-beta mRNA level was lowest in the SqCC/Y1 cells, and RXR-beta and RXR-gamma were not detected in any of the cell lines. RA treatment increased the levels of the three RAR mRNAs in most of the cell lines but had no effect on the RXR mRNAs. The CRABP-II mRNA level in SqCC/Y1 cells was lowest in cells grown in serum-free medium and increased when the cells were grown in medium with 5 or 10% serum. In contrast, the RXR-alpha mRNA level was inversely related to serum concentration. The results show that, in head and neck squamous cell carcinoma cells, there are no simple relationships among the expression of CRABPs, RARs, and RXRs and either squamous differentiation or response to RA-induced growth inhibition or suppression of squamous differentiation.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Head and Neck Neoplasms; Humans; Keratins; Protein Precursors; Receptors, Retinoic Acid; RNA, Messenger; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Expression of cytoskeletal proteins has been shown to be dependent on the differentiation status of the tissue. In the present study, expression of two cytoskeletal proteins normally present in terminally differentiated keratinocytes, namely cytokeratin types 10/11 and involucrin, were studied in different stages of tumour progression in the oral mucosa. Results showed that cytokeratins 10/11 and involucrin strongly correlated with the differentiation status of cells. High expression was observed in non-dysplastic hyperplastic epithelium as compared to normal, dysplastic and neoplastic epithelium. In addition, various grades of dysplasia showed an inverse correlation with expression of these proteins. Statistical analysis of the results also showed a negative correlation between the differentiation of upper spinal cells and the stage of tumour progression. It therefore appears that the proteins studied may be useful as markers for epithelial carcinogenesis.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Humans; Immunoenzyme Techniques; Keratinocytes; Keratins; Leukoplakia, Oral; Mouth Mucosa; Mouth Neoplasms; Protein Precursors

The expression of cytokeratins and involucrin varies greatly in different epithelia, and this raises the possibility that detailed analysis of these epidermal proteins might provide a means of identifying various skin tumours. The present study was conducted to determine the immunohistochemical distribution of cytokeratins and involucrin in calcifying epithelioma of Malherbe, in order to elucidate the nature and differentiation of this tumour. To correlate the immunohistochemical profile with the most frequent histological patterns, we categorized the basophilic, transitional, shadow, and squamoid cells, and the shreds of keratin. Comparative studies with normal skin showed that the shadow and transitional cells corresponded to hair cortex cells, the squamoid cells to the outer root sheath, the basophilic cells adjacent to the stroma to the outermost cell layer of the outer root sheath between the lower permanent portion and upper transient portion of the follicles, and the basophilic cells adjacent to the transitional cells to the hair matrix. The expression of cytokeratins in most shreds of keratin was similar to that in squamoid cells. Calcifying epithelioma was, therefore, shown to be composed of tumour cells differentiating into both the hair cortex and outer root sheath. These tumour cells were differentiated from basophilic cells, which showed the same staining patterns as the outermost cell layer of the outer root sheath between the lower permanent portion and upper transient portion of the hair follicles, supporting the hypothesis that the keratinocytes in the outermost cell layer can differentiate into the transitional portion of the follicle and anagen hair.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Hair Diseases; Humans; Immunohistochemistry; Infant; Keratins; Middle Aged; Pilomatrixoma; Protein Precursors; Skin; Skin Neoplasms

Topics: Bandages, Hydrocolloid; Biomarkers; Colloids; Epidermis; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratins; Occlusive Dressings; Protein Precursors; Psoriasis; Skin

The mode of differentiation of seborrhoeic keratoses was investigated by immunohistochemical staining using cytokeratin (CK) polypeptide-specific monoclonal antibodies and an antibody specific for the particulate form of epidermal transglutaminase (ETgase), and by applying an anti-human involucrin serum. The role played by (E)Tgase was further evaluated using an activity assay based on the covalent attachment of monodansylcadaverine. Samples of uninvolved epidermis served as reference tissue. CK reactivities suggested that seborrhoeic keratoses is a hyperproliferative disease with an epidermal CK composition. CK5 and CK14 were prominent markers of basal and basaloid keratinocytes, whereas a decrease in staining occurred in advanced maturation stages and areas of terminal keratinization. In contrast, CK1 and CK10 were prominent markers of suprabasaloid differentiation stages and produced complementary stainings to those of CK5 and 14. Generally, CK10 staining was more impressive than CK1 staining and seemed to start before CK1 staining. In contrast to CK10 staining, cornified areas lost CK1 reactivity. These staining patterns were similar to those observed in uninvolved reference tissues. The epidermal CK subset was further supplemented with the 'hyperproliferative' CK6 and 16 which occur sequentially. Positive staining for CK6 was noted from basal and proximal basaloid cells onwards, whereas distal basaloid cells additionally showed CK16 staining. The presence of other non-epidermal CK polypeptides could not be shown. The competence for other differentiation markers belonging to the group of (E)Tgase and cornifying cell membranes also evolved with a typical epidermal pattern. (E)Tgase activity was restricted to advanced and terminal stages of keratinization and was dual in nature, i.e. a diffuse cytoplasmic staining occurred together with a prominent staining of cornifying cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Humans; Immunohistochemistry; Keratins; Keratosis, Seborrheic; Protein Precursors; Transglutaminases; Vimentin

Whether the peripheral ameloblastoma (PA) and intraoral basal cell carcinoma (BCC) are two different clinical entities or essentially the same lesion still remains unresolved. The immunophenotypes of neoplastic cells of peripheral and intraosseous ameloblastomas, ameloblastic carcinomas, and BCCs were studied using a panel of monoclonal/polyclonal antibodies and lectins. The major cytokeratins (CKs) of neoplastic cells of ameloblastomas were CKs 5 and 14, whereas co-expression of CKs 8, 18, and 19 was observed in the cells of the stellate reticulum-like areas. Metaplastic squamous and keratinizing cells found in follicular and acanthomatous variants of ameloblastomas expressed CKs 1 and 10, involucrin, and binding sites for the lectins Ulex europeaus agglutinin I and Helix pomatia agglutinin. beta 2-Microglobulin was uniformly negative in all cases of ameloblastomas and ameloblastic carcinomas studied. Cutaneous BCCs also demonstrated similar reactive patterns with the above-mentioned antigens. The most striking feature is the presence of a peritumorous band-like peanut agglutinin staining found in both BCCs and PAs but not in intraosseous ameloblastomas. This unique peanut agglutinin staining pattern of PA may be diagnostically useful for its histopathologic distinction from an intraosseous ameloblastoma that has infiltrated the soft tissue. The neoplastic cells of ameloblastomas express markers of less-differentiated epithelial cells. Despite differences in epithelial origins, PAs are tumors analogous to cutaneous BCCs.

Topics: Adolescent; Adult; Aged; Ameloblastoma; Antigens, Differentiation; beta 2-Microglobulin; Bone Neoplasms; Carcinoma, Basal Cell; Female; Humans; Immunoenzyme Techniques; Keratins; Lectins; Male; Middle Aged; Protein Precursors; Receptors, Mitogen; Skin Neoplasms; Soft Tissue Neoplasms

To examine whether the tumor suppressor gene p53 influences epidermal differentiation, primary cultures of human foreskin keratinocytes and six human papillomavirus (HPV)-positive cell lines were infected with recombinant retroviruses encoding wild-type p53. Overexpression of p53 in organotypic cultures of normal keratinocytes decreased their growth rate and induced premature cell flattening and involucrin expression, a marker of squamous differentiation. However, overexpression of p53 inhibited or delayed production of other epidermal proteins, keratin 10, profilaggrin, and keratinocyte transglutaminase. Furthermore, levels of endogenous cellular p53 dramatically decreased during epidermal differentiation, suggesting that down-regulation of p53 permits complete expression of specific epidermal proteins. Three HPV-immortalized keratinocyte cell lines and three HPV-positive cervical carcinoma-derived cell lines expressed significantly less (< 3-fold) p53 protein than normal keratinocytes. Up-regulation of wild-type p53 (> 5-fold) by retrovirus infection did not significantly inhibit growth or restore normal epithelial differentiation in any line. Thus, overexpression of wild-type p53 can either induce or inhibit expression of specific epidermal proteins in normal keratinocytes but does not reverse immortality or aberrant differentiation of HPV-immortalized or carcinoma-derived cell lines.

Topics: Cell Differentiation; Cell Division; DNA, Viral; Filaggrin Proteins; Gene Expression; Genes, p53; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Papillomaviridae; Protein Precursors; Reference Values; Transfection; Tumor Cells, Cultured

Calcium is an important regulator of normal human cervical cell differentiation; however, it is not known whether the calcium responsiveness of the cells would be altered by immortalization with human papillomavirus. In the present study we examine the effects of extracellular calcium on morphology, expression of biochemical markers of cervical cell differentiation, and HPV16 oncogene transcription in an HPV16-immortalized human cervical cell line, ECE16-1. ECE16-1 cells respond to increased calcium with increased levels of mRNA encoding cytokeratin K13, transglutaminase, and involucrin. Dose-response studies indicate that these markers are coordinately regulated and that maximal levels are present at calcium concentrations of > or = 0.4 mM. RNA levels begin to increase within 24 h after cells are shifted to medium containing high calcium and are maximal by 3 days. Protein levels change directly with the change in mRNA, suggesting that mRNA level determines protein concentration; however, transcription runoff experiments indicate that the increased mRNA does not result from new synthesis. Parallel studies indicate that expression of the HPV16 oncogenes, E6 and E7, are not regulated by extracellular calcium. These results are consistent with in vivo experiments showing that in advanced cervical intraepithelial neoplasia, where HPV16 DNA is integrated in host cell DNA, E6/E7 transcript production is not regulated in a differentiation-dependent manner.

Topics: Calcium; Cell Differentiation; Cell Transformation, Viral; Cervix Uteri; Epithelial Cells; Epithelium; Female; Gene Expression Regulation, Viral; Humans; In Vitro Techniques; Keratins; Oncogene Proteins, Viral; Oncogenes; Papillomaviridae; Papillomavirus E7 Proteins; Protein Precursors; Repressor Proteins; RNA, Messenger; Transglutaminases; Tumor Virus Infections

The expression of cytokeratins (CK) 1, 4, 5/6, 8, 13, 18, 19 and 20 and involucrin in 42 cases of squamous cell carcinomas from various locations was examined. The tumours expressed CK5/6 in 55%, CK8 in 76%, CK13 in 43% and CK19 in 95% of cases. The CK5/6-positive primary tumours were from uterine cervix, head and neck, lung, skin, oesophagus and urinary bladder, and the CK13-positive primary tumours were from uterine cervix, lung and vulva. Metastatic squamous cell carcinomas from head and neck more frequently expressed CK5/6 and 13, 7/7 (100%) and 6/7 (86%) compared with 3/5 (60%) and 0/5 (0%) in the primary squamous cell carcinomas. Few cases were CK1, CK4 and CK18 immunoreactive. CK20 immunoreactivity was not observed. Involucrin was expressed in 71% of tumours, and most of the involucrin-positive cells were located at the central parts of tumour cell clusters except for one case in which the peripheral cells around tumour cell clusters were positive. Thus, expression of the so-called simple epithelial markers CK8 and CK19 occurs in the majority of squamous cell carcinomas. The absence of CK20 immunoreactivity may be helpful in differential diagnosis.

Topics: Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymphatic Metastasis; Mouth Neoplasms; Protein Precursors; Skin Neoplasms; Urinary Bladder Neoplasms; Uterine Neoplasms

The expression of involucrin, a structural component of the envelope of mature squamous epithelium, was studied in 166 paraffin-embedded breast carcinomas. In 41 cases (24.7%) involucrin-positive, light microscopically non squamous tumour cells were detected. The number of involucrin-positive tumour cells varied considerably from case to case. For further characterization, involucrin-positive cases were studied using monoclonal antibodies to various cytokeratins (PKK1, EAB 903, EAB 904) and, in selected cases, double immunostaining with antibodies to cytokeratins and involucrin were performed. Coexpression of involucrin and cytokeratins demonstrated by PKK1 was seen in all tumour cells, whereas coexpression of involucrin and cytokeratins detected by EAB 904 was only seen in single and scattered cells in a few cases. Cytokeratins detected by EAB 903 were not coexpressed with involucrin in our cases. Our results indicate heterogeneity of cytokeratins in breast carcinomas and suggest a dissociation in the regulation of involucrin and cytokeratin expression.

Topics: Breast Neoplasms; Carcinoma; Female; Humans; Immunohistochemistry; Keratins; Male; Protein Precursors

Extramammary Paget disease appears in anogenital, axillary, or other areas. In this study, the authors addressed the question of whether the histogenesis of 35 cases of Paget disease arising at different sites was the same.. Specimens of 35 cases of extramammary Paget disease (16 genital; 9 invasive carcinomas of genital; 6 axillary; 1 periumbilical; and 3 perianal), 4 cases of mammary Paget disease, 4 cases of breast carcinomas, and 6 cases of anal carcinomas of perianal spread from primary rectal adenocarcinomas were retrieved and stained by the avidin-biotin-complex method, using various kinds of monoclonal antikeratin antibodies.. There was no significant difference in cytokeratin expression among these cases of extramammary Paget disease. Simple epithelial keratins were expressed in Paget cells in extramammary Paget disease, but no expression of differentiation-specific or noncornifying stratified squamous epithelial keratins was observed, regardless of the degree of invasion. Paget cells in extramammary Paget disease revealed a similar cytokeratin expression to that in secretory cells of normal apocrine or eccrine glands. In addition, there was no significant difference in cytokeratin expression in tumor cells among extramammary and mammary Paget disease, breast carcinomas, and anal carcinomas.. Cases of Paget disease arising at different locations could not be distinguished from each other based on cytokeratin expression. In addition, antikeratin antibodies against simple epithelial keratins were demonstrated to be more useful for the identification of Paget cells in the paraffin sections than were conventional antibodies, such as an antibody against carcinoembryonic antigen (CEA).

Topics: Antibodies, Monoclonal; Breast Neoplasms; Carcinoembryonic Antigen; Carcinoma; Eccrine Glands; Gene Expression Regulation, Neoplastic; Humans; Keratins; Neoplasm Invasiveness; Paget Disease, Extramammary; Paget's Disease, Mammary; Protein Precursors; Urogenital Neoplasms

The immunocytologic characteristics of two formalin-fixed, paraffin-embedded corneas from patients with the iridocorneal endothelial (ICE) syndrome and unaffected control corneas were studied. Binding of polyclonal antisera to Factor VIII, S-100 protein, involucrin, neuron specific enolase (NSE), and the lectins peanut agglutinin and Ulex europaeus agglutinin-1 was performed using the standard peroxidase-anti-peroxidase method. We detected reactive patterns of monoclonal antibodies to cytokeratins (34BE12 is a 56-58 kD mouse IgG reactive to stratified epithelia; Pkk1 is a 44-54 kD mouse IgG reactive to simple epithelia; and KL1 is a 55-57 kD mouse IgG reactive to epidermis and simple epithelia) using the standard avidin-biotin complex method. Staining properties were similar for the polyclonal antisera, lectins, NSE, and chromogranin in corneas with ICE syndrome and in the controls. However, the cytokeratins 34BE12, Pkk1, and KL1 were detected in the endothelium of the corneas with the ICE syndrome but not in the controls. These findings suggest that various cytokeratins are expressed in the corneal endothelium in the ICE syndrome that are not expressed in unaffected corneal endothelium.

Topics: Antibodies, Monoclonal; Corneal Diseases; Endothelium, Corneal; Factor VIII; Humans; Immunoenzyme Techniques; Iris Diseases; Keratins; Microscopy, Electron, Scanning; Protein Precursors; S100 Proteins; Syndrome

The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum. It is made by cross-linking precursor proteins by a membrane-associated transglutaminase. Using the cornified envelope of cultured human keratinocytes as the immunogen, we obtained a number of monoclonal antibodies which stained epidermis in a variety of ways. The peripheral staining pattern has been associated with several envelope precursors and this has been confirmed by western blots. A mouse IgM monoclonal antibody directed against epidermal basal cell hemidesmosomes was also discovered. By immunofluorescence, the monoclonal antibody produced a strong linear staining of the basement membrane zone and a polar cap on trypsin-dissociated epidermal basal cells. By immunoelectron microscopy, immunoreactants were present in the attachment plaques of hemidesmosomes on guinea pig esophagus. However, no protein reactive with the antibody was detected. This study suggests that an antigen associated with the basal cell hemidesmosomes may be incorporated in the cornified envelope.

Topics: Animals; Esophagus; Fluorescent Antibody Technique; Guinea Pigs; Humans; Keratinocytes; Keratins; Male; Microscopy, Immunoelectron; Protein Precursors; Proteins

In normal epidermis, as previously reported, the first signs of differentiation occur within the basal layer in a subpopulation of keratinocytes that start to express K1 and K10 "supra-basal" keratin transcripts (20-30% of the basal cells) and proteins (5-10% of the basal cells). We found that in psoriatic lesions, the basal layer was devoid of cells expressing these early differentiation markers. This was already the case at the periphery of the lesions, where epidermis, although slightly acanthotic, still completes the keratinization process. In the center of the lesions, not only the basal layer, but also several rows of suprabasal cells, were negative for keratin K10 transcripts or protein. Moreover, the upper nucleated layers of involved epidermis were also devoid of K10 keratin transcripts or proteins. In normal epidermis, as previously reported, transcripts for the "basal" K5 keratin were mainly restricted to the basal layer, whereas the protein persisted in a few suprabasal layers. We found that in psoriatic epidermis, K5 keratin transcripts persisted in several suprabasal layers up to the level where K10 keratin transcripts appeared. These data, although not contradictory with previous reports showing a reduction of K1-K10 keratins and other differentiation markers in psoriasis, demonstrate that these quantitative changes are in fact the result of major qualitative differences in the distribution of these markers in psoriatic versus normal skin. Our results indicating that the onset of differentiation is delayed in psoriasis show that, contrary to conclusions accepted so far, not only the suprabasal compartment, but also the basal one, is abnormal in psoriatic epidermis.

Topics: Cell Differentiation; Epidermis; Humans; Keratins; Protein Precursors; Psoriasis; RNA Probes; RNA, Messenger

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.

Topics: Antigens, Neoplasm; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Humans; Keratins; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Retinoids (vitamin A analogues) inhibit the squamous differentiation of normal and malignant epithelial cells. This study investigated the ability of the head-and-neck squamous-cell carcinoma (HNSCC) cell line 1483 to undergo squamous differentiation in the absence and presence of beta-all-trans retinoic acid (RA). The growth of these cells in culture is accompanied by an increase in keratinocyte transglutaminase, involucrin and keratin KI, 3 established markers of squamous cell differentiation. Higher levels of these differentiation markers were detected in cells cultured in delipidized serum (DLS), from which endogenous retinoids have been extracted, than in cells cultured in fetal bovine serum (FBS), which contains retinoids. Treatment with I microM RA decreased the levels of the various differentiation markers in cells cultured in either FBS or DLS as revealed by immunofluorescent labelling of permeabilized cells and by immunoblotting of cell extracts using specific monoclonal or polyclonal antibodies. The cells' ability to cross-link proteins to form envelopes under the plasma membrane was stimulated in the presence of calcium ionophore but inhibited by RA. These results indicate that the malignant 1,483 HNSCC cells recapitulate the main characteristics of normal squamous-cell differentiation in culture and that RA suppresses this differentiation as it does in normal keratinizing epithelial cells.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Immunoblotting; Ionophores; Keratinocytes; Keratins; Neoplasm Proteins; Protein Precursors; Retinoids; Transglutaminases; Tretinoin; Tumor Cells, Cultured

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.

Topics: Adult; Antigens, Surface; Biomarkers; Cell Communication; Cell Differentiation; Epidermal Cells; Epidermis; Fibroblasts; Filaggrin Proteins; Fluorescent Antibody Technique; Hair; Humans; Intermediate Filament Proteins; Keratins; Laminin; Microscopy, Electron; Middle Aged; Organelles; Phenotype; Protein Precursors; Scalp; Skin; Skin Physiological Phenomena

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Autoantibodies; Cell Differentiation; Epidermis; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Protein Precursors; Psoriasis; Skin

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.

Topics: Cross Reactions; Cytoskeletal Proteins; Desmogleins; Desmoplakins; Desmosomes; Electrophoresis, Polyacrylamide Gel; Epidermis; Filaggrin Proteins; Fluorescent Antibody Technique; Foot; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Microscopy, Immunoelectron; Protein Precursors

Porokeratoses are known to give rise to squamous and basal cell carcinomas. In this study, we examined 15 lesions of porokeratosis immunohistochemically for evidence of aberrant keratinization using several markers of keratinocyte (KC) maturation and differentiation, including involucrin, filaggrin, cytokeratins, and the growth activation marker psi-3. The staining patterns obtained were compared with several non-premalignant parakeratotic skin lesions including psoriasis, pityriasis rosea, pityriasis rubra pilaris, irritated seborrheic keratosis, atopic dermatitis, seborrheic dermatitis, and verruca vulgaris. The centers of porokeratoses stained in a pattern identical to that observed in other premalignant keratinocytic lesions including actinic keratoses, recessive dystrophic epidermolysis bullosa, and nonhealing wounds. KCs beneath the cornoid lamella (CL) stained in a pattern similar to that observed in squamous cell carcinomas. KCs peripheral to the CL in the epidermis showed a normal staining pattern. The control non-premalignant parakeratotic lesions displayed a variety of staining patterns, but none showed a pattern identical to that observed in porokeratosis. The failure of KCs in porokeratoses to mature and differentiate normally may be related to the increased incidence of carcinomas associated with these lesions.

Topics: Epidermis; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratosis; Protein Precursors; Skin Neoplasms

Skin lesions of three patients with inflammatory linear verrucose epidermal naevus (ILVEN) were examined. Histologically, orthokeratosis and parakeratosis were alternately seen in the acanthotic epidermis. By N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide staining, the horny cells in the parakeratotic epidermis showed a cytoplasmic SH pattern and a weak membranous SS pattern. The orthokeratotic epidermis revealed an increased involucrin expression, whereas the parakeratotic epidermis showed almost no involucrin expression. Ultrastructurally, in the parakeratotic epidermis, the living keratinocytes had prominent Golgi apparatuses and vesicles in the cytoplasm. In the intercellular spaces in the upper spinous layer through to the lower horny layer, an electron dense, homogeneous substance was deposited. The cytoplasm of the horny cells was filled with keratin filaments and contained remnants of nucleus and cytoplasmic membrane structures, and some lipid droplets. The marginal band formation was incomplete. Most of these ultrastructural abnormalities were not found in the orthokeratotic epidermis. There are both similarities and differences in histopathogenesis of the parakeratotic epidermis between ILVEN and psoriasis. A unique finding was the lack of involucrin expression in the ILVEN parakeratotic epidermis.

Topics: Adult; Child; Child, Preschool; Female; Humans; Immunohistochemistry; Keratins; Male; Nevus; Protein Precursors; Skin; Skin Neoplasms

Discoid lupus erythematosus lesions show hyperkeratosis and atrophy, which may reflect abnormal epidermal proliferation, differentiation, or both. In this investigation, markers for epidermal proliferation, differentiation and inflammation were studied in cutaneous lesions of discoid lupus erythematosus. Frozen sections of biopsy specimens from 20 patients were examined immunohistochemically regarding Ki-67 staining and keratin 16 expression (parameters for proliferation), and the expression of keratin 10, involucrin, and filaggrin (parameters for differentiation). The inflammatory infiltrate was characterized with the use of antibodies against T lymphocytes, monocytes/macrophages, and Langerhans cells. With these markers, epidermal proliferation was found to be increased in discoid lupus erythematosus. Keratin 10 expression, a marker for early differentiation, showed the pattern of normal skin. Involucrin and filaggrin, markers for terminal differentiation, were expressed already in the lower part of the stratum spinosum, whereas in normal skin these markers were restricted to the stratum granulosum and the upper layers of the stratum spinosum, and the stratum granulosum and stratum corneum, respectively. Infiltrate analysis revealed the well-established picture. We conclude that in cutaneous lesions of discoid lupus erythematosus, hyperproliferation is combined with normal early differentiation and premature terminal differentiation of keratinocytes.

Topics: Adult; Aged; Cell Differentiation; Cell Division; Cell Nucleus; Epidermis; Female; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Langerhans Cells; Lupus Erythematosus, Discoid; Macrophages; Male; Middle Aged; Monocytes; Protein Precursors; Skin; T-Lymphocytes

Human epidermal keratinocytes, immortalized by the introduction of SV40-adenovirus recombinants (9), were maintained in a low calcium (0.15 mM) defined medium. When differentiation was induced by a higher extracellular calcium concentration (1.5 mM), these cells altered in shape and developed stratification with formation of desmosomes, while they demonstrated limited terminal keratinization. The expression of involucrin, one of the precursor proteins of the cornified envelope, became elevated as the cell density increased, but was not affected by calcium. These results indicate that the SV40 immortalized human keratinocytes, maintained in a low calcium defined medium, partially respond to changes in extracellular calcium concentration.

Topics: Calcium; Cell Count; Cell Differentiation; Cell Division; Cell Line, Transformed; Culture Media, Serum-Free; Flow Cytometry; Humans; Keratinocytes; Keratins; Microscopy, Electron; Protein Precursors; Simian virus 40

Epidermolysis bullosa represents a grouping of inherited skin diseases characterized by epidermal fragility and frequently wounded skin. The recessive dystrophic subtype of epidermolysis bullosa (RDEB) is characterized by extensive dermal scarring after healing of repeated epidermal injuries and by an unusually high incidence of squamous cell carcinoma occurring in chronically wounded skin. In contrast, the simplex form of epidermolysis bullosa usually heals without scarring and does not predispose to malignant neoplasms of the skin. The differences in scarring and the neoplastic potential of these two forms of epidermolysis bullosa prompted us to investigate growth activation and differentiation characteristics in epidermal keratinocytes in individuals with these disorders. The expression of filaggrin, involucrin, cytokeratins, and the growth activation marker psi-3 was examined by immunohistochemistry in skin biopsy specimens from four individuals with epidermolysis bullosa simplex and six individuals with RDEB. Previous experiments using this technique have demonstrated that these antibodies are good markers for identifying growth-activated keratinocytes in wounded and hyperplastic epidermis. All biopsy specimens of healed wounds in skin from patients with RDEB showed epidermis that reacted with antibodies to filaggrin, involucrin, specific cytokeratins, and psi-3 in a growth-activated pattern. This growth-activated phenotype was maintained in keratinocytes from previously wounded skin that had been healed for more than 2 years. The RDEB growth-activated phenotype detected by immunohistochemistry was not associated with microscopically detectable epidermal hyperplasia. In contrast, all cases of epidermolysis bullosa simplex examined showed an epidermal phenotype similar to that of keratinocytes in normal skin. Thus, healing with dermal scar formation in RDEB is associated with a persistent growth-activated immunophenotype of epidermal keratinocytes. This chronic growth activation state or failure of cells to differentiate in a normal fashion may be directly linked to the high incidence of squamous cell cancers in individuals with RDEB.

Topics: Adolescent; Adult; Antigens; Carcinoma, Squamous Cell; Epidermis; Epidermolysis Bullosa; Female; Filaggrin Proteins; Humans; Infant; Infant, Newborn; Intermediate Filament Proteins; Keratinocytes; Keratins; Male; Middle Aged; Protein Precursors; Retrospective Studies; Skin Neoplasms

While some cutaneous squamous cell carcinomas (SCC) arise from predisposing conditions such as burn scars, draining sinuses, and chronic, nonhealing wounds, the vast majority of these tumors arise from actinically damaged epidermis. It has been shown previously that keratinocytes within healing wounds show an "activated" immunophenotype when stained with antibodies to psi-3, involucrin, filaggrin, and cytokeratins. A similar pattern has been seen in keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB), in whom the incidence of cutaneous SCC is markedly increased. We tested the hypothesis that actinic keratoses (AK), recognized as precursors in the development of the majority of SCC, would show a similar activated immunophenotype when stained with the antibody panel described above. We examined 10 AK, biopsied from the facies and extremities of ten patients, ages 60 to 80, with antibodies to psi-3, involucrin, filaggrin, and AE1. All lesions examined had an immunostaining pattern indistinguishable from that seen in keratinocytes from patients with RDEB or within healing wounds. There was suprabasilar staining of keratinocytes with antibodies to psi-3 and AE1. Involucrin and filaggrin was expressed by all keratinocytes above the midstratum spinosum. Within the acrosyringia and acrotrichia, the staining pattern was that of the normal epidermis, i.e., AE1 staining of basal keratinocytes, granular layer staining of involucrin and filaggrin, and absence of psi-3 expression. These data suggest that an activated keratinocyte phenotype is a unifying feature in conditions which predispose to development of cutaneous SCC.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epidermis; Female; Filaggrin Proteins; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratosis; Male; Middle Aged; Phenotype; Precancerous Conditions; Protein Precursors; Skin Neoplasms; Sunburn

Human papillomavirus (HPV) DNAs are detected in approximately 90% of anogenital carcinomas. To assess directly the effect of HPV on squamous differentiation, normal human cervical and foreskin epithelial cells and cells immortalized by recombinant HPV DNAs were transplanted beneath a skin-muscle flap in athymic mice. Xenografts containing normal cells formed well-differentiated stratified squamous epithelia 2 to 3 weeks after transplantation, but cell lines immortalized by four HPV types (HPV16, HPV18, HPV31, and HPV33) detected in anogenital cancer exhibited dysplastic morphology and molecular alterations in gene expression characteristic of intraepithelial neoplasia. Morphological alterations were accompanied by delayed commitment to terminal differentiation, alterations in the pattern of involucrin expression, and reductions in levels of involucrin and keratin 1 RNAs. HPV18-immortalized cells developed dysplastic changes more rapidly than cells immortalized by HPV16 DNA. These results show that human genital epithelial cells immortalized by HPV DNAs detected in genital cancers undergo dysplastic differentiation in vivo.

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Line; Cell Transformation, Viral; Cervix Uteri; Epithelium; Female; Gene Expression Regulation, Viral; Genes, Viral; Humans; Keratins; Laminin; Male; Mice; Mice, Nude; Papillomaviridae; Penis; Protein Precursors; RNA; Transfection

Keratoderma striatum (Brünauer-Fuhs type) with linear keratotic elevations on the palms and small islets (areata form) on the soles is a rare form of palmoplantar keratoderma (PPK). An immunohistochemical and ultrastructural study has been performed to characterize the altered keratinization and maturation patterns in this disease before and during complete clinical remission on therapy with etretinate. Anticytokeratin antibody KL1 showed no significant difference in reaction pattern either between healthy controls and PPK or following therapy. Earlier expression of both filaggrin and involucrin was found in PPK in comparison with the controls. During etretinate therapy the filaggrin pattern returned to normal, whereas the altered involucrin pattern was not influenced. Ultrastructural investigations before treatment revealed tightly packed tonofibrils (TF) and large masses of keratohyalin (KH) granules with abnormal configuration. During therapy the TF and KH granules were reduced in number and size. KH granules now showed frayed borders. Moreover, a transitional cell zone, focal parakeratosis with lipid droplets, and dyskeratotic cells became apparent. The normalization of filaggrin pattern accompanying the clinical remission of these lesions implies a role of this keratinocyte differentiation protein in the pathogenesis of these lesions. Since etretinate is assumed to act at a very late stage of epidermal differentiation, there was no influence on the altered expression of involucrin during etretinate therapy. Despite the clinical remission, fine structural abnormalities persisted, indicating that the deviations from the normal keratinocyte differentiation program in PKK occur very early.

Topics: Adult; Cell Differentiation; Cell Division; Cytoplasmic Granules; Epidermis; Etretinate; Filaggrin Proteins; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratinocytes; Keratins; Keratoderma, Palmoplantar; Male; Microscopy, Electron; Protein Precursors; Staining and Labeling

The well and poorly differentiated oral squamous carcinomas preferentially express proteins, blood group antigens, and contain associated dendritic Langerhans' cells. Keratin pearls in well-differentiated carcinomas simulate the differentiation pathway of the normal oral squamous epithelium, whereas poorly differentiated carcinomas do not and appear more heterogeneous. Terminally keratinized cells correlate with involucrin and expression of blood group antigens in keratin pearls, a feature that differs from the nonkeratinizing normal epithelium in which such carcinomas arise. Dendritic Langerhans' cells are reduced in number in squamous carcinomas.

Topics: ABO Blood-Group System; beta 2-Microglobulin; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Dendritic Cells; Fibronectins; HLA Antigens; Humans; Keratins; Laminin; Langerhans Cells; Mouth Neoplasms; Protein Precursors; S100 Proteins

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Drug Evaluation; Female; Humans; Immunoenzyme Techniques; Isotretinoin; Keratins; Male; Micronucleus Tests; Middle Aged; Mouth Neoplasms; Protein Precursors; Transglutaminases

A histochemical study was performed to clarify further the role played by epidermal transglutaminase (ETgase) in the keratinization of aural cholesteatoma. Weakly and strongly keratinized epidermal tissues and healthy middle ear mucosa were included as references. A first assay revealed the distribution of non-specified acyl donor substrates. In a second assay, the topography of involucrin was assessed immunohistochemically. In both epidermal and cholesteatoma matrix tissues, the presence of acyl donors was not restricted to the sites of (E)Tgase activity, but was almost uniformly extended throughout living layers. In reference tissues, residual acyl donors were poorly detected in horny layers, while they were more abundant in the stratum corneum of the cholesteatomas studied. The presence of involucrin along the cell membrane was observed at varying distances throughout the spinous and granular layers, depending upon the epidermal and matrix configurations. In thick epithelia, involucrin rapidly became concentrated at the cell periphery (in spinous keratinocytes), while in thin epithelia it was usually associated with cell flattening. This latter staining profile was observed more frequently in cholesteatomatous tissues. In addition, we regularly noticed an immediately suprabasal accumulation of involucrin, suggesting a locally hyperproliferative state of the matrix. An insufficient availability of acyl donors, especially involucrin, could not be used to explain the defective ETgase-mediated cross-linking of cholesteatoma cell membranes during terminal stages of differentiation. The present investigation may be the first to demonstrate the presence of involucrin in middle ear mucosa.

Topics: Cholesteatoma; Ear Diseases; Ear, Middle; Humans; Immunoenzyme Techniques; Keratins; Mucous Membrane; Protein Precursors; Transglutaminases

Immortalized, but nontumorigenic, human keratinocyte cell lines have many potential therapeutic and experimental uses. We have utilized a recombinant retrovirus, encoding the simian virus 40 large T-antigen, to immortalize normal human epidermal keratinocytes. The KER-1 cells derived from the immortalization process grow without feeder layer support, but do not form colonies in soft agar. Morphologically, the KER-1 cells appear similar to nonimmortalized cells, except that stratification is somewhat reduced. The pattern of keratin gene expression in nonimmortalized and KER-1 cells is similar, except for the retinoid-dependent regulation of type I cytokeratin, K7, in the KER-1 cells. This keratin is not expressed in nonimmortalized keratinocytes, but is present at low levels in KER-1 cells. Incubation with trans-retinoic acid (20 or 200 nM) or retinol (200 or 2000 nM) results in a 40-fold increase in K7 expression in KER-1 cells. The cornified envelope precursor, involucrin, is expressed at normal levels in KER-1 cells. Moreover, as in nonimmortalized cells, KER-1 involucrin levels are not suppressed by retinoids. trans-Retinoic acid and retinol reduce envelope formation in both nonimmortalized keratinocytes and KER-1 cells. Surprisingly, the synthetic retinoid, Ro 13-6298 (p [(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthyl)-propenyl]benzoic acid ethyl ester), a potent regulator of keratin gene expression, cornified envelope formation and morphological change in nonimmortalized cells, is completely inactive in KER-1 cells.

Topics: Antigens, Polyomavirus Transforming; Cell Transformation, Viral; Gene Expression Regulation; Humans; Keratinocytes; Keratins; Molecular Weight; Protein Precursors; Retinoids

We studied the proliferation and differentiation of human laryngeal papillomas, which are benign tumors induced by human papillomaviruses. Immunofluorescent stains of tissues for a number of differentiation-specific proteins showed abnormal differentiation. Papilloma tissue fragments in vitro showed a slightly decreased fraction of proliferating cells that incorporated tritiated thymidine and a markedly reduced incorporation of tritiated uridine when compared with normal tissue. We propose that papillomavirus infection results in normal basal cell proliferation but abnormal terminal differentiation and that this abnormality significantly contributes to the hyperplasia of the papillomas.

Topics: Cell Division; Cell Transformation, Neoplastic; Filaggrin Proteins; Humans; Immunoblotting; Intermediate Filament Proteins; Keratins; Laryngeal Neoplasms; Neoplasm Proteins; Papilloma; Papillomaviridae; Protein Precursors; Staining and Labeling; Thymidine; Tumor Virus Infections; Uridine

A fully differentiated epithelium having the features of epidermis was obtained in vitro by culturing second-passage normal human keratinocytes (NHK) in the chemically defined medium MCDB 153 on inert filter substrates at the air-liquid interface for 14 d. Vertical sections stained for histology and indirect immunofluorescence studies show a correct stratification and expression of differentiation markers. The presence of desmosomes, keratohyalin granules, and lamellar granules, and the formation of a more than ten-layers stratum corneum was evidenced by electron microscopy. Moreover, lipids typical for differentiated epidermis were present in these cultures, including ceramides, which are thought to be responsible for the relative impermeability of the stratum corneum. Under our culture conditions, i.e., in defined medium and at the air-liquid interface, the use of de-epidermized dermis as a substrate did not stimulate keratinocyte differentiation more than acetate cellulose or polycarbonate filter membrane substrates. The obtaining of a well-differentiated epidermis grown in vitro on inert filters in a chemically defined medium should be useful as a standard system for studying epidermal differentiation, re-epidermization, cytotoxicity, epidermal permeation, and transepidermal drug delivery.

Topics: Adult; Air; Antibodies, Monoclonal; Cell Differentiation; Cells, Cultured; Culture Media; Epidermal Cells; Epidermis; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratinocytes; Keratins; Microscopy, Electron; Protein Precursors; Transglutaminases

In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host-mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs.

Topics: Adolescent; Adult; Aged; Cell Differentiation; Cell Separation; Cells, Cultured; Child; Electrophoresis, Gel, Two-Dimensional; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Keratinocytes; Keratins; Male; Mesoderm; Middle Aged; Protein Precursors; Skin; Skin Transplantation

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Epidermal Cells; Humans; Hydrocortisone; Keratins; Protein Precursors; Transglutaminases; Tretinoin

The authors have studied the expression of keratin 19 in normal oral mucosa and in oral lesions exhibiting a range of histopathologic changes that are thought to precede squamous cell carcinoma. Formalin-fixed, paraffin-embedded sections were pretreated with pronase and stained with a K19-specific antibody by the avidin-biotin immunoperoxidase method. In nonkeratinized mucosa, whether normal or benign hyperplastic, K19 was detectable in the basal cell layer. In keratinized mucosa, whether normal or benign hyperplastic, there was no detectable K19. All lesions from any oral site that exhibited atypia diagnosed from hematoxylin and eosin stained sections as moderate-to-severe dysplasia or carcinoma in situ, whether hyperkeratotic or not, stained strongly for K19 in the basal and suprabasal cell layers. The number of cell layers that were K19-positive correlated with the level in the epithelium to which dysplasia persisted. Suprabasal K19 staining tended to occur in regions of the epithelium in which expression of the terminal differentiation protein involucrin was delayed or absent. Thus, K19 expression may be linked to the retention of stem cell character or a state otherwise uncommitted to terminal squamous differentiation. Suprabasal K19 staining is clearly correlated with premalignant change in oral epithelium and therefore promises to be a useful tool in oral histopathologic diagnosis.

Topics: Epidermis; Humans; Immunohistochemistry; Keratins; Molecular Weight; Mouth Mucosa; Precancerous Conditions; Protein Precursors

Cryostat sections of human skin were stained with monoclonal antibodies to involucrin, a range of cytokeratins, epithelial membrane antigen (EMA), and an ovarian cystadenocarcinoma antibody (OM1) to identify combinations of antibodies that could be used to discriminate between basal and differentiated sebocytes and other cell types present in the pilosebaceous unit. Both the EMA and OM1 monoclonal antibodies specifically recognized differentiated sebocytes. No staining of basal sebocytes or other epidermal cell types was seen. Differentiated (but not basal) sebocytes were also stained by a cytokeratin 10 antibody (LH2). Conversely, the basal sebocytes were recognized by an antibody specific to basal keratinocytes (LH6). Cells of the sebaceous duct stained with both LH2 and LH6 and also with the anti-involucrin monoclonal antibody. Cytokeratin 4 has been detected in sebaceous glands by protein analysis but has not previously been detectable immunohistochemically. We show by immunofluorescence after limited proteolysis that cytokeratin 4 epitopes are distributed in all sebaceous gland cells, including the duct cells.

Topics: Antibodies, Monoclonal; Antigens, Surface; Biomarkers; Cell Differentiation; Cell Membrane; Epithelium; Humans; Immunohistochemistry; Keratins; Mucins; Protein Precursors; Sebaceous Glands

The protein involucrin is a precursor of the cross-linked envelope that forms during terminal differentiation of the keratinocyte. Most of the human involucrin molecule consists of a segment of homologous repeats of a sequence of 10 amino acids. A similar segment is present in the involucrin of other higher primates, but not in lower animals. We show here that the older part of the involucrin molecule (the ancestral segment) is present in the epidermal cells of subprimates. This has been demonstrated with antisera prepared against different peptides of the ancestral segment of the human protein. No single antiserum detects involucrin of all subprimate species, but probably all involucrins can be detected using antiserum against some sequence in the ancestral segment. Although the involucrin gene has been extensively remodeled in higher primates, its origins extend lower in the animal kingdom.

Topics: Amino Acids; Animals; Dogs; Epidermal Cells; Epidermis; Immune Sera; Keratins; Lemur; Membrane Proteins; Protein Precursors; Rabbits; Transglutaminases

Undifferentiated cervical carcinomas vary considerably in their intercellular organization and patterns of invasion. In spite of its clinical significance, the basis for such variation is poorly understood. We investigated the cellular properties that may be responsible for this diversity, using as a model two human cervical carcinoma cell lines that were derived from the same tumor specimen and the same clone. It was shown previously that, in spite of their common origin, each line forms a histologically distinct type of undifferentiated carcinoma when heterotransplanted in vivo: cells of line C-4I grow as compact expanding masses with central necrosis, while tumors of line C-4II infiltrate host tissues as small, well-vascularized, dispersed cell groups. The characteristic behavior of each line was retained in culture, where C-4I cells formed highly multilayered cohesive colonies, while C-4II cells formed diffuse, monolayered colonies and shed into the culture medium. These observations as well as ultrastructural data suggested that each line may be arrested at a different stage of stratified squamous differentiation. In the present study, this hypothesis was tested by examining specific differentiation markers. An analysis of the cultures by immunofluorescence microscopy and immunoblotting revealed that keratin was more abundant in the compact C-4I line than in the dispersed C-4II line. C-4I cells expressed keratins 5, 6, 8, 16, 18, and 19, while C-4II expressed only keratins 8, 16, 18, and 19. In the multilayered C-4I colonies, involucrin-positive cells occurred in the apical cell layers only. In C-4II, involucrin-positive cells occurred in monolayers and domes, and they were most consistently located apically in crowded cultures. Laminin was secreted by both lines, but only C-4II cells deposited a fibronectin matrix. The results suggest that C-4I cells resemble normal cervical cells at the spinous stage of stratified squamous differentiation, while C-4II cells resemble basal/suprabasal cells. The different growth patterns of the tumors, formed by the lines in vivo, therefore likely reflect functional and behavioral differences that normally exist between spinous and basal cervical epithelial cells. The results suggest that differentiation-related functional properties may lead to histological diversity among cervical carcinomas that are categorized as undifferentiated by histopathological criteria.

Topics: Carcinoma; Cell Differentiation; Female; Fibronectins; Humans; Keratins; Laminin; Neoplasm Proteins; Protein Precursors; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Hyper- and hypovitaminosis A both provoke epithelial pathologies in animals and humans. This suggests that a critical level of retinoic acid (RA) is required in vivo for the maintenance of normal architecture and function of these tissues. However, no beneficial, but only adverse effects of RA on epithelia have been so far observed in vitro. For instance, addition of RA to keratinocyte cultures has been shown to inhibit epidermal differentiation while this process is stimulated by serum delipidization, which reduces RA concentration in the medium. Assuming that the previous failure to demonstrate beneficial effects of RA on the epidermal phenotype in vitro was due to culture conditions too far from the in vivo conditions we decided to reevaluate the effect of RA in a culture system optimized for epidermal morphogenesis: the "emerged dermal equivalent." When human keratinocytes were grown in such a system with total fetal calf serum, the resulting epithelium was very similar to normal epidermis. But when delipidized serum was used, the epithelium was abnormal in the direction of excessive maturation (hyperkeratosis). When physiological concentrations of RA (10(-9) and 10(-8) M) were added to the delipidized serum supplement, a normal architecture (orthokeratosis) was restored. However, as classically described in the literature, higher RA concentrations (greater than 10(-7) M) reduced epidermal maturation and produced parakeratosis. Thus, although it is unquestionable that RA reduces the synthesis of epidermal-specific differentiation markers, an optimal epidermal morphogenesis seems to be achieved only in the presence of a critical RA concentration.

Topics: Cell Differentiation; Cell Line; Culture Media; Epidermis; Epithelium; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Lipids; Morphogenesis; Protein Precursors; Receptors, Fibronectin; Receptors, Immunologic; Transglutaminases; Tretinoin

We examined 20 punch biopsies taken from five patients at varying intervals following CO2 laser-induced thermal injury. The regenerating epidermis was studied with monoclonal antibodies AE1 and AE3 (directed against low and high molecular weight keratins), and involucrin, a protein found within the cellular envelope of the most mature keratinocytes. Twenty-four hours following thermal damage, there was extensive spillage of keratins and involucrin into the papillary dermis and disarray of all constituents of the necrotic keratinocytes. Early ingrowth of basaloid keratinocytes weakly expressed AE1. By 1 week, keratinocytes expressed AE1 in varying intensities throughout the epidermis. AE3 was present in its normal distribution, staining all but the most basaloid keratinocytes. Involucrin stained cells deep within the epidermis. Six weeks following the initial injury, the staining pattern within the epidermis had returned to normal. Thus, it appears that the regenerating epidermis produces low molecular weight keratins in cells at all levels and forms premature cellular envelopes, perhaps as a protective measure, before expression of these constituents reverts to the normal pattern. These findings suggest that keratinocyte differentiation in wound-healing following laser-induced thermal injury is similar to that seen in other types of injury. Observed clinical differences may be attributable to differences in keratinocyte proliferative or migratory capabilities.

Topics: Adult; Cell Differentiation; Epidermis; Humans; Keratins; Laser Therapy; Middle Aged; Protein Precursors; Regeneration; Wound Healing

In the epidermis proliferation of keratinocytes is restricted to the basal layer, which is in contact with the basement membrane, and cells undergo terminal differentiation as they move upwards through the suprabasal layers. In stratified cultures of human keratinocytes, upward migration is a consequence, not a cause, of terminal differentiation and occurs because keratinocytes become less adhesive to their substratum and to one another. Most keratinocytes can be induced to differentiate to completion by placing them in suspension in methylcellulose: within 12 h DNA synthesis is irreversibly inhibited and by 24 h most cells express involucrin (ref 4; P. A. Hall, J.C.A. and F.M.W., unpublished observations). Here we report that when fibronectin is added to the methylcellulose, keratinocytes still withdraw from the cell cycle, but induction of involucrin expression is largely inhibited. The effect of fibronectin is concentration- and time-dependent and is mediated by a receptor of the integrin family. These results provide an explanation for why overt terminal differentiation is normally restricted to suprabasal cells, whereas cell-cycle withdrawal occurs within the basal layer; they also have important implications for the mechanism of epidermal wound healing. Furthermore, our data show that the binding of an extracellular matrix protein to its receptor can regulate differentiated gene expression in the absence of changes in cell shape.

Topics: Cell Differentiation; Epidermal Cells; Epidermis; Fibronectins; Humans; In Vitro Techniques; Keratins; Protein Precursors; Receptors, Fibronectin; Receptors, Immunologic; Serum Albumin, Bovine; Thymidine; Transforming Growth Factors

Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin. This is due in part to difficulties in culturing human fetal epidermal keratinocytes. We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates. To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation. Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins. However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane. More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts, including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins. The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis.

Topics: Antibodies, Monoclonal; Basement Membrane; Cell Aggregation; Cell Differentiation; Cells, Cultured; Collagen; Culture Media; Epidermal Cells; Epidermis; Fluorescent Antibody Technique; Histocytochemistry; Humans; Immunoblotting; Keratins; Laminin; Molecular Weight; Protein Precursors; Skin; Vimentin

The distribution of several markers of keratinocyte differentiation was studied in normal epidermis, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs) using the immunoperoxidase technique on frozen sections of punch biopsy specimens. As markers a panel of chain-specific monoclonal antibodies (MoAbs) directed against cytokeratin (CK) 4, 8, 10, 13, 18 and 19, a polyclonal antiserum against involucrin, as well as a MoAb against the epidermal growth factor (EGF) receptor were used. In 15 out of 19 BCCs tested, expression of CK 8 was seen. Only a few individual cells in a limited number of BCCs showed positive staining for CK 4, 18, or 19. No expression of CK 10 was seen except for some foci of cell keratinization. Involucrin was not found in BCCs except for some squamous horn cysts. In all BCC cells expression of EGF receptor was found. In the suprabasal layers of normal epidermis from SCC patients, positive staining for CK 10 was seen. A few individual cells in a limited number of SCCs showed positive staining for CK 4, 8, or 18. Involucrin was expressed in the center of SCCs and in the upper layers of normal epidermis. Expression of EGF receptor was found in all SCC cells. These results demonstrate differences in cellular origin and differentiation between BCC and SCC.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermal Cells; Epidermis; ErbB Receptors; Humans; Immunoenzyme Techniques; Keratins; Protein Precursors; Skin Neoplasms

Cell line MDA 886Ln was established from a laryngeal lymph node metastasis. When grown as a multicellular tumor spheroid (MTS), it exhibits squamous differentiation. We studied the effects of beta-all-trans retinoic acid (RA) on the growth, differentiation and glycoprotein content of this MTS model for squamous carcinomas of the head and neck. The growth of MTSs was inhibited in a dose-dependent manner by 10(-6) to 10(-10) M RA. Growth inhibition occurred between 3 and 5 days of RA treatment (10(-6)M). Immunohistochemical and electrophoretic analyses revealed that RA suppressed the morphological markers of squamous differentiation (squames), involucrin expression, and keratin expression. Gly-coprotein expression was examined by metabolic labelling using 3H-glucosamine, in situ labelling of polyacrylamide gels with 125I-labelled wheat-germ agglutinin (WGA), localization of fluorescein isothionate-WGA in frozen sections, and determination of sialyltransferase activity. Treatment using 10(-6) M RA altered glycoprotein expression both biochemically and morphologically, and WGA was shown to bind preferentially to sialic acid residues. The sensitivity of this MTS model to RA treatment and its ability to be analyzed through morphological, immunohistochemical and biochemical techniques suggest that it will prove useful in studying the relationships between growth, differentiation and RA-induced alterations in squamous carcinomas.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Glycoproteins; Head and Neck Neoplasms; Humans; Keratins; Male; Molecular Weight; Neoplasm Proteins; Organoids; Protein Precursors; Tretinoin; Tumor Cells, Cultured

As the distribution pattern of cytokeratin (CK), filaggrin and involucrin has recently been suggested to discriminate between benign and malignant epithelial growths, biopsies of healthy oral mucosa, leukoplakias without and with dysplasia and squamous cell carcinomas were examined immunohistochemically using a panel of 4 monoclonal antibodies (AB) against different cytokeratin polypeptides (34 beta E12, KL1 and Pkk1) and filaggrin as well as a polyclonal AB to involucrin. Major and statistically significant differences were observed in the profiles of CKs (except Pkk1), filaggrin and involucrin between leukoplakias without and with epithelial dysplasia. However, the alteration in the expression of CKs, filaggrin and involucrin proved to be not a constant feature in leukoplakias with dysplasia as a considerable portion (20-25%) of them revealed the profiles of CKs, filaggrin and involucrin similar to those of benign leukoplakias, and vice versa. Immunostaining of these antigens did not define the diagnosis of dysplasia in leukoplakias more precisely than grading in conventional histology can do so far. However, immunohistochemical sensitivity in detecting a broad range of variation in the abnormal maturation patterns of keratinocytes in leukoplakias with dysplasia can be used to divide these lesions into subgroups to elucidate their prognosis in follow-up studies.

Topics: Carcinoma, Squamous Cell; Epithelium; Female; Filaggrin Proteins; Humans; Hyperplasia; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratins; Leukoplakia, Oral; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Phosphoproteins; Protein Precursors

A comparison of normal epithelial cells with their transformed counterparts could lead to the definition of parameters related to growth and differentiation which are altered by viral transformation and which may be relevant to malignant changes in vivo. Using the SV40-transformed human keratinocyte line, SVK14, which exhibits characteristics of simple, nonkeratinizing epithelia, we have shown that IGF I stimulation of these cells results in extensive multilayering, increased cell size, accumulation of involucrin, modulation of keratin 18 and expression of keratins 14 and 10, whilst T-antigen expression is maintained in the multilayered cells. Since T-antigen expression is correlated directly with impairment of stratification and differentiation, it is interesting that treatment of SVK14 with a single growth factor. IGF I, results in molecular events characteristic of differentiating normal keratinocytes.

Topics: Antigens, Viral, Tumor; Cell Differentiation; Cell Division; Cell Line, Transformed; Flow Cytometry; Growth Substances; Humans; Immunoblotting; Insulin; Insulin-Like Growth Factor I; Keratinocytes; Keratins; Protein Precursors; Simian virus 40; Somatomedins

The epithelia lining the cyst of five cases of calcifying odontogenic cyst (COC) were evaluated immunohistochemically with the use of monoclonal antibodies (MoAb's) against keratin (PKK1, KL1, K4.62, K8.12) and vimentin, and polyclonal antisera agonist involucrin and filaggrin. Epithelial lining of COC was classified into 1) thin squamous-cell epithelium, 2) ameloblastoma-like, and 3) thin or 4) thick calcifying odontogenic epithelium. Foci consisting of ghost cells or calcified cells were categorized as calcifying epithelial odontogenic tumor (CEOT). Thin squamous-cell epithelium reacted with PKK1, KL1, K4.62, K8.12, and anti-vimentin MoAb's, thus demonstrating the co-expression of keratin and vimentin. Ameloblastoma-like cells showed positive staining with PKK1, KL1, and sometimes with anti-vimentin. Thick calcifying odontogenic epithelial lining showed stratification of cell layers, and the most strikingly reactive zone was the upper intermediate layer, which showed the presence of keratin, involucrin, and a small amount of filaggrin. Cells of this layer might be the most differentiated type of cells in COC. Undifferentiated odontogenic cells of COC masses were characterized by co-expression of keratin and vimentin, and by the absence of involucrin and filaggrin. All ghost cells were devoid of any immunostaining except for filaggrin, which was rarely positive, but eosinophilic or basophilic cells surrounding the ghost cells showed intense staining for all keratin proteins except vimentin.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Epithelium; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Jaw Diseases; Keratins; Male; Odontogenic Tumors; Protein Precursors

Calcifying odontogenic cysts (COC) were immunohistochemically described using different keratin proteins and involucrin as well as histopathology. The cystic lining epithelium was composed of calcifying, keratinizing, squamous, and columnar epithelial cells, and included calcified masses of irregular shape and various size as well as ghost cells. Calcifying epithelium gave negative or only trace staining for keratins detected with low molecular keratin (PKK1), but were regularly positive with high molecular keratin (KL1) and polyclonal antibody for keratin (TK). They were occasionally positive for involucrin. The cells located in the periphery of the calcified masses had a particular abundance of high molecular weight and total keratins (KL1 and TK). Calcified bodies and ghost cells were devoid of any immunoreactivity. Squamous epithelium was relatively similar to that of normal squamous cell epithelium in the oral mucosa. It were most commonly found in columnar cystic epithelial cells which displayed intense staining with all immunoreagents. It is postulated that such epithelial cells may have a strong potentiality to transform into ghost cells or to undergo metaplasia. They may develop altered synthesis of homogenous acellular materials and finally become transformed into calcifying epithelium containing dystrophic calcified masses.

Topics: Adolescent; Adult; Child; Epithelial Cells; Female; Gingival Neoplasms; Humans; Immunohistochemistry; Keratins; Male; Odontogenic Tumors; Protein Precursors

Forty nasopharyngeal carcinomas (NPC) were studied by immunohistochemistry using an antibody to involucrin and the following three keratin antibodies: (1) an antibody to low molecular weight keratin reactive with nonsquamous epithelium, (2) a high molecular weight keratin antibody reactive with suprabasal squamous epithelium, and (3) a keratin antibody reactive with full thickness stratified epithelium. In its pattern of reactivity, the last antibody overlaps the low and high molecular weight keratin antibodies and is used as a broad spectrum keratin antibody. By World Health Organization (WHO) classification, the cases in this article included eight keratinizing squamous cell carcinomas, eight nonkeratinizing carcinomas, 20 undifferentiated carcinomas, and four adenocarcinomas. The antibody to broad spectrum keratin had an overall sensitivity of 87.5% and was positive in all eight keratinizing squamous cell carcinomas, seven nonkeratinizing carcinomas (87.5%), 18 undifferentiated carcinomas (90%), and two adenocarcinomas (50%). Low molecular weight keratin antibody stained one additional NPC, which was negative when broad spectrum keratin antibody was used. Involucrin and high molecular weight keratin antibodies demonstrated near parallel staining in all histologic classes; there was marked localization to areas of squamous differentiation. While involucrin is a marker for foci of greater squamous differentiation, broad spectrum keratin antibody may aid in the diagnosis of all histologic subtypes of NPC.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Antibody Specificity; Carcinoma; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Nasopharyngeal Neoplasms; Protein Precursors

Two cases of clear cell acanthoma are reported. The expression of carcinoembryonic antigen (CEA), involucrin and keratin proteins in the tumors was investigated immunohistochemically. In 1981, Penneys et al. reported that this tumor was not of sweat gland origin because of the absence of CEA. This study confirmed this, further, the pattern of positive reaction of involucrin also indicated that this tumor is not of sweat duct origin.

Topics: Aged; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Protein Precursors; Skin Neoplasms

Human foreskin keratinocytes in culture produce 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) from 25-hydroxycholecalciferol (25-(OH)D3). The production of 1,25-(OH)2D3 by these cells correlated with the early events of differentiation such as expression of transglutaminase activity and the levels of a precursor protein for the cornified envelopes, involucrin. In contrast, the increased production of 24,25-(OH)2D3, as 1,25-(OH)2D3 production declined, correlated with the terminal differentiation marker, cornified envelope formation. Exogenous 1,25-(OH)2D3 (10(-11)-10(-9) M) inhibited the 1-alpha-hydroxylase at all stages of growth of these cells. Keratinocytes in culture expressed receptors for 1,25-(OH)2D3 which had similar sedimentation behavior in sucrose density gradients as chick intestinal cytosol receptors. Cells in early stages of growth (preconfluent and confluent) contained higher numbers of receptors (26-27 fmol/mg protein) than post-confluent cells. The dissociation constant (237-278 pM) of these receptors for 1,25-(OH)2D3 was not consistently altered by differentiation. Since 1,25-(OH)2D3 is a potent stimulator of cell differentiation in a variety of systems including the epidermis, our results suggest the possibility that endogenous 1,25-(OH)2D3 production may participate in the differentiation of keratinocytes in culture and, perhaps, in vivo.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Cell Differentiation; Chickens; Dihydroxycholecalciferols; Epidermal Cells; Humans; Intestinal Mucosa; Keratins; Microscopy, Electron; Protein Precursors; Receptors, Calcitriol; Receptors, Steroid; Transglutaminases

In normal epidermis there is a balance between the rate of cell division and the rate of terminal differentiation. This can be perturbed by injuries such as wounding or tape-stripping, but is reestablished during recovery. The aim of the studies described in this report was to assess whether cultures of human keratinocytes could be used as an experimental model for investigating the mechanism by which epidermal homeostasis is established and maintained. The suprabasal layers were stripped from confluent keratinocyte cultures by incubation in low-calcium medium (0.1 mM calcium ions). After return to normal medium (2 mM calcium ions), the basal layer regenerated a stratified culture of approximately the same thickness as controls. The kinetics of proliferation and terminal differentiation were monitored by measuring the total number of cells and proportion of involucrin-positive cells at intervals before, during, and after stripping. During recovery the proportion of cells expressing involucrin, assessed by immunofluorescence microscopy and polyacrylamide gel electrophoresis, rapidly returned to control levels, but the total number of cells per dish rose more slowly and often failed to reach control values. Thus, terminal differentiation was initially stimulated at the expense of proliferation. Our in vitro model of epidermal regeneration should provide a useful complement to intact skin and animal models for analyzing epidermal homeostasis.

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Cytological Techniques; Epidermal Cells; Epidermis; Homeostasis; Humans; Infant, Newborn; Keratins; Kinetics; Male; Protein Precursors

Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.

Topics: Animals; Calcium; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cells, Cultured; DNA, Viral; Epithelial Cells; Epithelium; Gene Expression Regulation; Immunologic Techniques; Keratins; Male; Mice; Mice, Nude; Neoplasms, Experimental; Papillomaviridae; Protein Precursors; RNA, Messenger; RNA, Viral

Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture.

Topics: Cell Adhesion; Cell Communication; Cell Differentiation; Cell Division; Cells, Cultured; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epidermis; Fluorescent Antibody Technique; Humans; Keratins; Microscopy, Electron, Scanning; Protein Precursors

In the human squamous carcinoma cell line SCC-9, the expression of two markers of keratinocyte differentiation, involucrin and transglutaminase, was greatly stimulated when growing cultures reached confluence. However, the two markers differed temporally in their induction, with transglutaminase reaching maximal levels shortly after confluence and involucrin a week later. If replication was arrested with hydroxyurea prior to confluence, transglutaminase induction occurred within several days but involucrin levels were completely suppressed. Such a striking degree of uncoupling also resulted when the cells were treated with polycyclic aromatic hydrocarbons such as benzo[a]pyrene but not with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, or with pyrene. Chronic treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed expression of both transglutaminase and involucrin. However, suppression of the latter (evident in greatly reduced mRNA levels) was considerably more potent and powerful. These findings demonstrate uncoupling of keratinocyte differentiation, potentially useful in analysis of its multiple regulatory influences. They also emphasize the utility of sensitive keratinocyte targets for studying the mechanisms by which model carcinogens disturb the orderly progression of events in their differentiation program.

Topics: Benzo(a)pyrene; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Enzyme Induction; Epidermal Cells; Epidermis; Humans; Hydroxyurea; Keratins; Kinetics; Protein Precursors; Tetradecanoylphorbol Acetate; Transglutaminases

We have characterized an unusual cell phenotype in third passage cultures of a human keratinocyte strain derived from newborn foreskin epidermis. The cells had the same DNA fingerprint pattern as the second passage, morphologically normal, keratinocytes; they formed desmosomes and expressed the keratin profile characteristic of normal keratinocytes in culture. However, unlike normal keratinocytes, the cells did not grow as compact colonies and did not stratify or undergo terminal differentiation, even after TPA treatment or suspension culture. For these reasons we named them ndk for "nondifferentiating keratinocytes." The ndk cells also differed from normal keratinocytes in that they did not require a feeder layer and were not stimulated by cholera toxin to proliferate. The ndk cells had an absolute requirement for hydrocortisone and their growth rate was increased when epidermal growth factor was added to the medium. Although ndk failed to undergo terminal differentiation in culture, they were not transformed, since they were still sensitive to contact inhibition of growth, did not proliferate in soft agar, and had a limited lifespan in culture. The appearance of the ndk phenotype was correlated with a doubling of chromosome number and the presence of a lp marker chromosome. We suggest that these cells are a useful experimental adjunct to cultures of normal keratinocytes, in which proliferation and terminal differentiation are tightly coordinated, because in ndk cells there appears to be a block in terminal differentiation.

Topics: Blotting, Western; Cell Differentiation; Cell Division; Cells, Cultured; Cytoskeleton; Desmosomes; DNA Probes; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Humans; Karyotyping; Keratins; Phenotype; Ploidies; Precipitin Tests; Protein Precursors; Skin

The protein involucrin, synthesized by human keratinocytes, contains 585 amino acids, largely in the form of 10 amino acid repeats, each containing glutamines in 3 conserved positions. Involucrin is a substrate for the keratinocyte transglutaminase and is labeled by the cosubstrate amine, glycine ethyl ester. Study of tryptic peptides of involucrin shows that a single glutamine (residue 496), located 89 residues from the C-terminal end, is preferentially labeled by the enzyme. Additional glutamine residues become reactive when the molecule is fragmented. The C-terminal end, isolated as a cyanogen bromide fragment of 275 residues, is labeled equally at 2 glutamine residues. The polypeptide containing residues 148 to 280 accepts practically no amine while in intact involucrin but as a free fragment is labeled at multiple glutamine residues. It is concluded that the C-terminal and N-terminal ends of the protein are directive influences in that they suppress the reactivity of a number of glutamine residues in the intact molecule, leaving one glutamine highly preferred by the transglutaminase.

Topics: Amino Acid Sequence; Cells, Cultured; Cross-Linking Reagents; Cyanogen Bromide; Cytosol; Epidermis; Glutamine; Humans; Keratins; Peptide Fragments; Protein Precursors; Transglutaminases

Twenty-one cases of Paget's disease have been studied using histochemical, ultrastructural, and immunohistochemical methods. Eight of the tumors involved the nipple, and 13 were extramammary (11 vulvar and two anal). The antibodies used were directed against different classes of cytokeratin proteins, epithelial membrane antigen, carcinoembryonic antigen, gross cystic disease fluid protein-15, and S-100 protein. The findings of this study provide conclusive evidence that Paget's cells, regardless of their location, are adenocarcinoma cells. Intracytoplasmic mucin is scanty in Paget's cells within the nipple, but typically plentiful in the extramammary sites where the cells are frequently signet-ring cells. The common mechanism for the evolution of Paget's disease is extension of cells from an underlying carcinoma, but the possibility that some cases, particularly in the vulva, develop from intraepithelial precursors cannot be excluded.

Topics: Antibodies, Monoclonal; Anus Neoplasms; Apolipoproteins; Apolipoproteins D; Breast Neoplasms; Carcinoembryonic Antigen; Carcinoma, Intraductal, Noninfiltrating; Carrier Proteins; Female; Glycoproteins; Humans; Keratins; Membrane Proteins; Membrane Transport Proteins; Mucin-1; Paget Disease, Extramammary; Paget's Disease, Mammary; Protein Precursors; S100 Proteins; Vulvar Neoplasms

SV-40 transformed human foreskin keratinocytes (line SV-K14) develop under conditions of serum starvation the competence to form cornified envelopes that are characteristic of terminally differentiating epidermal cells. In this cell line, the final assembly of the envelope does not occur spontaneously but must be induced using a calcium ionophore. Five potential precursor proteins with molecular weights of 140K, 90K, 61K, 53K, and 36K, respectively, could be detected in the extracts of envelope competent and noncompetent cells. The 61 kD and the 36 kD precursors were specifically decorated in immunoblots when using an antiserum directed against the purified cornified envelope of SV-K14 cells. The 140 kD protein was identified as involucrin by means of a commercial anti-involucrin antibody. Part of the 61 kD protein was found to be inserted into the plasma membrane after the cells gained envelope competence. The set of precursor proteins used by SV-K14 cells differed markedly from those described in the literature for epidermal cells in vivo and for normal human keratinocytes in vitro. Furthermore, cyanogen bromide cleavage of purified envelopes from transformed and normal keratinocytes revealed a completely different peptide pattern. This indicates that the exact molecular composition of the cornified envelope may not be strictly determined and may vary according to the availability of potential substrate proteins at the very moment when the cross-linking enzyme, the plasma membrane associated transglutaminase, becomes functional.

Topics: Cell Line; Cell Transformation, Viral; Electrophoresis; Humans; Keratins; Male; Protein Precursors; Simian virus 40; Skin; Subcellular Fractions; Tissue Distribution; Viral Envelope Proteins

Eccrine syringofibroadenoma is a rare tumor considered to originate from the excretory portion of the eccrine sweat gland. A new case of this lesion, whose acrosyringeal differentiation was underlined by an immunohistological study using antibodies to keratin and involucrin, is reported herein.

Topics: Adenofibroma; Aged; Aged, 80 and over; Antibodies, Monoclonal; Diagnosis, Differential; Humans; Keratins; Male; Protein Precursors; Sweat Gland Neoplasms

Squamous cell carcinomas of the urinary bladder and the epithelial lesions associated with infection by Schistosoma haematobium were histopathologically and immunohistochemically described for keratin proteins (TK, 41-65 kDa; KL1, 55-57 kDa; PKK1, 40, 45 and 52.5 kDa), involucrin, and epithelial membrane antigen (EMA). Normal urothelial epithelium was positive for all keratins, and showed absent or slight reactions for involucrin and EMA in superficial umbrella cells. The intestinal type of epithelium was composed of columnar cells and small basal cells; TK was positive in the basal cells, KL1 staining was positive in the columnar cells, whereas PKK1 was negative or slight in the columnar cells. Involucrin was confined to columnar cells. Squamous metaplastic epithelium showed a rather regional keratin distribution: TK was distributed in all layers, KL1 decorated upper spinous and granular layers, but PKK1 did not bind, and involucrin staining existed only in upper spinous and granular cells. Keratin expression in squamous cell carcinomas indicated heterogeneity and its stainability was dependent on the degree of keratinization: The G 1 type revealed strong reaction, the G 2 type showed a similar distribution pattern, but the staining intensity was less, and the G3 type showed irregular staining with decreased intensity. Involucrin staining was limited to keratinized cells of carcinoma as was that for EMA.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Histocytochemistry; Humans; Immunochemistry; Keratins; Male; Membrane Proteins; Middle Aged; Mucin-1; Protein Precursors; Schistosomiasis haematobia; Urinary Bladder Neoplasms

Involucrin, the major protein precursor of the cornified envelope, is expressed during terminal differentiation of human keratinocytes, both in vivo and in vitro. In epidermis, the onset of synthesis is several layers above the basal layer, but in stratified cultures of keratinocytes on tissue culture plastic involucrin synthesis begins in the first suprabasal layer. To investigate the reason for this premature expression, the distribution of involucrin was studied in epidermis from different body sites, in organotypic cultures and in transplants of keratinocytes onto nude mice. We found that premature expression was not associated with poor morphological differentiation, because involucrin synthesis began immediately above the basal layer even when distinct basal, spinous, granular and cornified layers were formed in organotypic cultures recombined with dermis. The site of involucrin expression in culture did not depend on the number of cornified layers present. The only conditions which resulted in an upward shift in the site of synthesis were in 3-week old transplants on nude mice. We conclude that the site of onset of involucrin synthesis is not determined by the degree of morphological differentiation of the tissue, and discuss other factors which may be involved.

Topics: Adult; Animals; Cell Differentiation; Cells, Cultured; Epidermal Cells; Epidermis; Histocytochemistry; Humans; Keratins; Mice; Mice, Nude; Protein Precursors; Transplantation, Heterologous

Nasal mucosal biopsy specimens encompassing normal and pathological epithelia were obtained from nickel workers, a population with an increased incidence of carcinomas of the respiratory tract. The immunohistochemical detection of keratins was carried out using monoclonal antibodies AE1 and AE3. The antibody AE1 stained only the basal cells of normal mucociliary epithelium. Regular metaplasias showed an increased stain with AE3; all layers of the surface epithelium were stained. The dysplastic and neoplastic lesions exhibited an extraordinary increase in the staining patterns with AE3 and to a lesser extent with AE1. Involucrin, which was absent from the normal pseudostratified epithelium, appeared in all metaplastic-dysplastic lesions and in keratinized areas of carcinomas. The combined detection of keratins and involucrin proved useful in detecting the degree of maturity and differentiation of preneoplastic and neoplastic lesions of the nasal mucosa.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epithelium; Humans; Immunohistochemistry; Keratins; Microscopy, Electron; Nasal Mucosa; Nickel; Nose Neoplasms; Occupational Diseases; Precancerous Conditions; Protein Precursors

Topics: Cell Differentiation; Epidermal Cells; Epidermis; Humans; Keratins; Protein Precursors; Skin Diseases

The role of type beta transforming growth factor (TGF beta) and epidermal growth factor (EGF) as regulators of the growth and differentiation of cultured human neonatal epidermal cells and squamous carcinoma cells was investigated in postconfluent cultures. Neither cell proliferation nor DNA synthesis was affected by treatment with TGF beta alone; however, EGF significantly stimulated cell growth, and this process was specifically antagonized by TGF beta. In addition, TGF beta inhibited the maturation of human foreskin-derived epidermal cells, as measured by their competence to synthesize involucrin and to form cornified cell envelopes, in a dose-dependent manner. Although treatment with EGF did not affect the maturation of human foreskin-derived epidermal cells, the combination of a low concentration of TGF beta with EGF resulted in significant enhancement of the maturation of these normal keratinocytes. Growth of three of four squamous carcinomas in the presence of EGF was not inhibited by TGF beta. In addition, all four carcinomas were either totally or partially resistant to the induction of maturation by the combination of TGF beta and EGF. This resistance of squamous carcinomas to TGF beta was paralleled by an increased sensitivity to the antikeratinizing effects of EGF. Thus, TGF beta inhibited the mitogenic stimulation of keratinocytes by EGF and induces cell maturation.

Topics: Cell Differentiation; Cell Division; DNA Replication; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Keratins; Peptides; Protein Precursors; Transforming Growth Factors

A total of 211 cases of benign and malignant tumors of epithelial origin were studied by the immunoperoxidase method to determine the distribution profile of epithelial membrane antigen (EMA) with the use of monoclonal antibody. Normal epithelial cells in the oral mucosa and skin were usually negative for EMA staining, as were epithelial cells in hyperplastic lesions and papillomas. Paget cells and tumor cells of Bowen's disease (carcinoma in situ) demonstrated a high incidence of EMA positivity, whereas the frequency in basal cell carcinoma was unexpectedly low. Squamous cell carcinomas revealed positive EMA staining of cytoplasmic membranes, and the antigen was also present in keratinized areas. EMA expression in squamous cell carcinoma generally showed a high incidence (85%) and was higher in keratinized lesions than in unkeratinized or less well-differentiated neoplasms. EMA distribution could be classified into two forms: one in which the cytoplasmic membranes demonstrate positivity and in which a positive cytoplasmic pattern is found in parakeratinized cells in malignant foci.

Topics: Antibodies, Monoclonal; Antigens; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mouth Mucosa; Mouth Neoplasms; Mucin-1; Protein Precursors; Skin Neoplasms

When malignant human cells are crossed with diploid human keratinocytes, malignancy, as defined by progressive growth in vivo, is suppressed so long as the hybrid cells continue to produce involucrin, a protein that characterizes terminal differentiation in the keratinocyte. When, on continued cultivation in vitro, the cells lose the ability to produce involucrin, they reacquire the ability to grow progressively in the animal.

Topics: Cell Division; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epidermis; Humans; Hybrid Cells; Immunoenzyme Techniques; Keratins; Neoplasms; Protein Precursors

Two months after transplantation of human skin onto the nude mouse, excisional wounds were made through the entire thickness of the skin, at the center of the graft, using a 2-mm punch. At various time intervals thereafter, ranging from 2 days to 9 weeks, the graft sites were harvested and processed for an immunohistological study. With a monoclonal antibody directed against HLA-ABC antigens, it was demonstrated that the healing epidermis is of human origin. Moreover, with three different monoclonal antibodies directed against human keratins, named respectively AE1, AE3, and KL1 and with an anti-involucrin antiserum, it is reported that the keratinization and involucrin distribution patterns observed in normal human epidermis are reconstituted, 2 months after transplantation, in the major part of the grafted epidermis, undergo changes during the reepithelialization process, and are restored in the healed epidermis 9 weeks after injury. This study indicates that the nude mouse/human skin model could be a valuable tool to study a major aspect of regeneration such as the reepidermization of human skin without recourse to human volunteers.

Topics: Animals; Epidermis; Fluorescent Antibody Technique; Humans; Keratins; Mice; Mice, Nude; Protein Precursors; Skin; Skin Transplantation; Transplantation, Heterologous; Wound Healing

We examined seven invasive squamous cell carcinomas, five squamous cell carcinomas in situ, four keratoacanthomas, two actinic keratoses, and two seborrheic keratoses by indirect immunofluorescence. We used a panel of three antibodies: one directed against filaggrin, one against involucrin, and one against peptidylarginine deiminase. Anti-involucrin stained all the lesions studied, but the pattern within a given category of lesions was variable and consistent differences between the categories were not observed. Similarly, the antibodies against peptidylarginine deiminase and filaggrin were not able to distinguish differences between the various types of tumors. We conclude that in tumors of epidermis, benign or malignant, products of differentiation are expressed independently of histologic atypia or clinical aggressiveness. Therefore, markers of differentiation do not appear to be reliable indexes for distinguishing benign from malignant lesions.

Topics: Carcinoma, Squamous Cell; Filaggrin Proteins; Fluorescent Antibody Technique; Humans; Hydrolases; Intermediate Filament Proteins; Keratins; Keratoacanthoma; Precancerous Conditions; Protein Precursors; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Skin; Skin Neoplasms

Fifteen keratoacanthomas and fifteen squamous cell carcinomas of the skin were examined by immunoperoxidase methods for involucrin and both 45- and 63-kilodalton keratins. Keratoacanthomas showed a relatively homogeneous staining pattern for involucrin; all cells except basal cells stained with mild to moderate intensity. Squamous cell carcinomas disclosed a highly irregular involucrin staining pattern with marked variation in staining intensity from cell to cell. Staining patterns for keratin proteins did not appear to distinguish between keratoacanthomas and squamous cell carcinomas. The 45-kilodalton keratin pattern showed diffuse staining within both keratoacanthomas and squamous cell carcinomas, and the 63-kilodalton keratin pattern consisted of focal staining, mostly of dyskeratotic cells. These results suggest that involucrin may serve as a diagnostic aid in differentiating between squamous cell carcinomas and keratoacanthomas. In addition, other lesions in the differential diagnosis of keratoacanthoma and squamous cell carcinoma were also examined for involucrin.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Epidermis; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Keratoacanthoma; Middle Aged; Neoplasm Proteins; Protein Precursors; Sebaceous Glands; Skin Diseases; Skin Neoplasms; Sweat Glands

Involucrin has been recognized recently as a marker of terminal differentiation of squamous epithelial cells and also as a useful marker for keratinization; its expression in epithelial tumors of oral and pharyngeal mucosa and skin was examined. Involucrin in normal oral mucosa and skin was restricted to the granular and upper spinous layers and was absent in the basal layer. Hyperkeratosis was characterized by strong positive staining for involucrum in spinous and granular cell layers. A similar pattern was noted in seborrheic keratosis and verruca vulgaris. Condyloma acuminatum specimens revealed slight staining, whereas Paget cells were negative. Calcifying epitheliomas of Malherbe were usually unreactive. Papillomas exhibited the regular distribution of involucrin, as found in normal squamous epithelium. Basal cell carcinomas were generally negative, whereas squamous cell carcinomas showed an irregular distribution of involucrin. Immunohistochemical staining for involucrin may be useful for identification of keratinizing cells in epithelial tumor foci, just as is the use of monoclonal antibody to keratin KL1.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Mouth Diseases; Mouth Mucosa; Mouth Neoplasms; Papilloma; Pharyngeal Neoplasms; Protein Precursors; Skin Diseases; Skin Neoplasms; Staining and Labeling

Forty-seven surgically resected small cell lung carcinomas (SCLC) were immunohistochemically studied by using antibodies to various neuroendocrine and epithelial markers. SCLC was shown to be subdivided into two categories, with and without the immunoreactive neuroendocrine markers aromatic L-amino acid decarboxylase, gastrin-releasing peptide, serotonin, chromogranin A and neurofilament protein. Neuron-specific enolase (NSE) and creatine kinase BB isoenzyme (CK-BB), which are also considered to be neuroendocrine markers, had a tendency to be widely distributed in the SCLC with a neuroendocrine marker, but the immunoreactivity for both NSE and CK-BB varied in the SCLC without neuroendocrine markers. Therefore they were not included in the classification. Epithelial markers keratin, involucrin and epithelial membrane antigen were frequently observed in the SCLC with neuroendocrine markers, but less so in the SCLC without neuroendocrine markers. The data are discussed briefly in relation to "classic and variant" forms of SCLC in vitro and to a recently proposed histological classification of SCLC.

Topics: Aromatic-L-Amino-Acid Decarboxylases; Carcinoma, Small Cell; Creatine Kinase; Gastrin-Releasing Peptide; Histocytochemistry; Humans; Immunochemistry; Isoenzymes; Keratins; Lung Neoplasms; Neurosecretory Systems; Peptides; Phosphopyruvate Hydratase; Protein Precursors

The presence of keratins and involucrin was studied in 9 human fetuses of an age-range between 9 and 24 weeks of gestation. The use of the monoclonal antibodies AE1 and AE3 as well as an anti-involucrin serum enabled developmental changes in the oral epithelia to be assessed. Our study detected some evidence of fetal differentiation patterns different from the adult, i.e. presence of AE3-Positive basal cells and AE1-Positive suprabasal cells in early stages of fetal development. In addition, involucrin was detected as early as 9 weeks of gestation in the superficial cells of the oral cavity epithelium. After the 15th week of gestation the differentiation markers studied gradually adopted adult-type distribution patterns.

Topics: Antibodies, Monoclonal; Cell Differentiation; Epithelium; Fetus; Fluorescent Antibody Technique, Indirect; Gestational Age; Humans; Immunoenzyme Techniques; Keratins; Mouth; Protein Precursors

In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis, and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes. Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration dependence and stereospecificity for these responses suggest the involvement of the Ah receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating basal cells, modulating the responsiveness of these cells to growth factors such as EGF.

Topics: Cell Differentiation; Cell Line; Cells, Cultured; Clone Cells; Dioxins; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Keratins; Keratosis; Male; Polychlorinated Dibenzodioxins; Protein Precursors; Receptors, Aryl Hydrocarbon; Receptors, Drug

Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.

Topics: Cells, Cultured; Epidermal Cells; Epidermis; Epithelium; Humans; Immunochemistry; Keratins; Microscopy, Electron; Protein Precursors; Skin

Human lung tumor cell lines established from the major histological types of lung cancer were examined by immunofluorescent staining techniques for their patterns of intermediate filament (keratin, vimentin, and neurofilament triplet protein) expression. All cell lines examined, both small cell lung carcinoma (SCLC) and non-SCLC (squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and mesothelioma) contained keratin, consistent with their epithelial derivation. These lung carcinoma cell lines also expressed vimentin, the characteristic intermediate filament of mesenchymal cells in vivo. In light of the proposed neuroectodermal origin of SCLC, cell lines were also studied for neurofilament expression. Two of four SCLC tumor cell lines, as well as non-SCLC cell lines, showed no reactivity with antibodies to neurofilament triplet protein. Two of the SCLC cell lines stained weakly with anti-neurofilament antibody. Examination of specific keratin patterns in human lung tumor cell lines by selective immunoprecipitation with keratin antiserum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that small-sized keratin proteins (Mr 44,000 to 52,000) were present in cell lines derived from SCLC and non-SCLC types of lung cancer. Tumor cell lines exhibiting squamous differentiation by light microscopic criteria (i.e., intracellular keratin, intercellular bridging, "pearl" formation, and/or individual cell keratinization) also displayed a preponderance of intermediate-sized keratins (Mr 57,000 and 59,000) and exhibited another feature of terminal keratinocyte differentiation (cross-linked envelope formation). Mesothelioma cell lines had varying keratin profiles. The presence of keratin proteins in all SCLC cell lines examined argues against a neuroectodermal origin for these tumors and is consistent with the notion that these tumors arise from a common bronchial "stem cell," similar to that from which other types of bronchogenic carcinomas arise.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Lung Neoplasms; Mesothelioma; Mice; Microfilament Proteins; Molecular Weight; Protein Precursors; Vimentin

A transglutaminase-catalyzed cross-linking process characteristic of keratinocytes leads to the formation of the insoluble corneocyte envelope. The essentials of this process take place in vitro in a reconstituted system derived from subcellular fractions. A particulate fraction containing membrane-bound envelope precursor proteins and the enzyme transglutaminase is combined with cytosolic proteins; when the enzyme is activated by Ca++, cytosolic proteins are removed from solution and cross-linked to particulate proteins. This interaction is cell-type-specific, since particulates derived from fibroblasts and also containing transglutaminase activity cannot substitute for those of keratinocytes. Involucrin, a cytosolic protein known to be a precursor of the envelope, is more efficiently cross-linked than other cytosolic proteins. The cross-linking of proteins of the particulate fraction (membrane proteins) is promoted by the presence of involucrin.

Topics: Acyltransferases; Cell Membrane; Cells, Cultured; Cytosol; Epidermis; Humans; Keratins; Kinetics; Macromolecular Substances; Membrane Proteins; Protein Precursors; Putrescine; Transglutaminases

A study was undertaken to determine whether immunoperoxidase stains for keratin and involucrin, the latter a protein present in cells of stratified squamous epithelium that have differentiated beyond the basal stage, distinguish any differences in squamous cells present in the adenoacanthoma from those in the adenosquamous carcinoma of the uterine corpus. Forty-eight tumors were studied, of which 33 were adenoacanthomas and 15 adenosquamous carcinomas. The patients with adenoacanthomas were slightly younger (mean 61.5 vs. 64.5 years) and had tumors that were generally better differentiated than the adenosquamous carcinomas. The squamous epithelium in every tumor, regardless of histologic type, stained positively for keratin. There were no obvious differences in staining when tumors were stratified for histologic type, grade, or location within the tumor. The glandular portion of both tumor types stained irregularly, but nonetheless positively, for keratin in 71% of the cases. Involucrin was detected in 57% of adenoacanthomas and 87% of adenosquamous carcinomas. The deeper or more central portion of the squamous morules stained only if the more superficial or peripheral areas were positive. The extent of the involucrin staining was less in the adenosquamous carcinomas than in the adenoacanthomas. The glandular component of the tumors did not stain for involucrin. It is concluded that no qualitative differences in the staining reactions with respect to keratin and involucrin distinguish the adenoacanthomas from the adenoaquamous carcinoma. These findings support the argument that the adenoacanthoma and adenosquamous carcinoma represent a spectrum of squamous differentiation in a single tumor type.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Cell Differentiation; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Keratins; Middle Aged; Protein Precursors; Uterine Neoplasms

Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis.

Topics: Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Humans; Keratins; Molecular Weight; Protein Precursors; Simian virus 40; Skin

Involucrin is a precursor of cross-linked protein of human stratum corneum, and its appearance in the upper layers of the epidermis is a function of the normal differentiation of the keratinocyte. Cases of basal cell and squamous cell carcinoma were evaluated for the presence of involucrin using immunoperoxidase techniques on paraffin sections. Basal cell carcinomas were negative for involucrin with staining restricted to squamous horn cysts, while squamous cell carcinomas stained strongly, particularly in large keratinized cells. Cases of squamous cell carcinoma in situ (Bowen's disease) revealed increased staining for involucrin with staining of dyskeratotic cells at all levels in the epithelium. Abnormal patterns of staining were also noted in non-neoplastic epidermis adjacent to carcinomas. Immunohistochemical staining for involucrin identifying abnormal or premature keratinization is a sensitive marker for dyskeratosis in squamous epithelia and may have applications in the histopathologic evaluation of skin specimens.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermis; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Protein Precursors; Skin Neoplasms

Involucrin accumulation and ionophore-assisted envelope formation, markers of keratinocyte differentiation, were found to be highly dependent on culture conditions in the malignant epidermal keratinocyte line, SCC-13, derived from a human squamous cell carcinoma. In confluent cultures, approximately one-half of the cells were competent to form envelopes when grown in medium without hydrocortisone or retinyl acetate supplementation. Addition of hydrocortisone to the medium during growth resulted in up to 90% competence, while addition of retinyl acetate instead resulted in as low as 10% competence. Hydrocortisone partially antagonized the effect of retinyl acetate when both agents were added together. Involucrin levels, measured by radioimmunoassay, were modulated essentially in parallel with envelope competence under the various conditions tested. When the cells were grown in medium supplemented with hydrocortisone, the levels shortly after confluence were over 50-fold higher than in sparse cultures. Regardless of hydrocortisone or retinyl acetate addition, less than 1% of the cells were competent in sparse cultures of growing cells, but up to 90% exhibited this property after growth arrest in serum-free medium containing hydrocortisone. High levels of competence were correlated with cessation of cell division but not with loss of colony-forming efficiency; under optimal conditions, two-thirds of the cells were capable of both envelope formation and colony initiation. Normal human epidermal cells showed a 4- to 5-fold increase in envelope competence from sparse to confluent culture but were insensitive to the suppressive effect of retinyl acetate. The results suggest that some potential differentiated character of malignant keratinocytes may be suppressed in vivo by physiological agents such as vitamin A.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Membrane; Diterpenes; Humans; Hydrocortisone; Keratins; Protein Precursors; Radioimmunoassay; Retinyl Esters; Skin; Time Factors; Vitamin A

Based on recent morphologic, histochemical, and biochemical data, we propose a heterogeneous two-compartment model of the stratum corneum that ascribes a special role for intercellular lipids in the regulation of stratum corneum barrier function and desquamation. The evidence in favor of the model and several predictions based on the model are surveyed in this review.

Topics: Ceramides; Fatty Acids, Nonesterified; Histocytochemistry; Humans; Keratins; Lipid Metabolism; Lipids; Protein Precursors; Skin; Skin Diseases; Staining and Labeling; Sterols; Steryl-Sulfatase; Sulfatases