tretinoin and involucrin

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

Nuclear receptor-mediated signaling via RARs and PPARδ is involved in the regulation of skin homeostasis. Moreover, activation of both RAR and PPARδ was shown to alter skin inflammation. Endogenous all-trans retinoic acid (ATRA) can activate both receptors depending on specific transport proteins: Fabp5 initiates PPARδ signaling whereas Crabp2 promotes RAR signaling. Repetitive topical applications of ovalbumin (OVA) in combination with intraperitoneal injections of OVA or only intraperitoneal OVA applications were used to induce allergic dermatitis. In our mouse model, expression of IL-4, and Hbegf increased whereas expression of involucrin, Abca12 and Spink5 decreased in inflamed skin, demonstrating altered immune response and epidermal barrier homeostasis. Comprehensive gene expression analysis showed alterations of the cutaneous retinoid metabolism and retinoid-mediated signaling in allergic skin immune response. Notably, ATRA synthesis was increased as indicated by the elevated expression of retinaldehyde dehydrogenases and increased levels of ATRA. Consequently, the expression pattern of genes downstream to RAR was altered. Furthermore, the increased ratio of Fabp5 vs. Crabp2 may indicate an up-regulation of the PPARδ pathway in allergen-induced dermatitis in addition to the altered RAR signaling. Thus, our findings suggest that ATRA levels, RAR-mediated signaling and signaling involved in PPARδ pathways are mainly increased in allergen-induced dermatitis and may contribute to the development and/or maintenance of allergic skin diseases.

Topics: Allergens; Animals; ATP-Binding Cassette Transporters; Dermatitis; Fatty Acid-Binding Proteins; Female; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Interleukin-4; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Ovalbumin; PPAR delta; Protein Precursors; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Serine Peptidase Inhibitor Kazal-Type 5; Serpins; Signal Transduction; Tretinoin

All-trans retinoic acid (RA) is known to regulate keratinocyte proliferation and differentiation, and retinoids are used as therapeutic agents in certain dermatological disorders, such as psoriasis and acne. Epidermal expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced by RA treatment and HB-EGF is responsible for RA-mediated epidermal hyperplasia in vivo. RA also induces HB-EGF expression in cultured keratinocytes and alters their differentiating phenotype. R115866 is a specific inhibitor of the cytochrome P450 isoform CYP26, which is involved in the metabolic inactivation pathway of RA. Thereby, R115866 is thought to be able to increase the intracellular levels of endogenous RA.. To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes.. We analysed HB-EGF, involucrin and keratin 10 mRNA and protein levels in autocrine human keratinocyte cultures incubated for 18 h with RA or R115866 alone and with RA and R115866 combinations.. RA induced HB-EGF and involucrin expression in a concentration-dependent manner, whereas it inhibited keratin 10 expression. R115866 alone had no effect on the expression of these genes. However, when R115866 was combined with low concentrations of RA, HB-EGF and involucrin expression was induced.. These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

Topics: Adult; Benzothiazoles; Cells, Cultured; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Epidermal Cells; Epidermis; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Keratin-10; Keratinocytes; Protein Precursors; Tretinoin; Triazoles

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca(2+) or 1alpha,25-dihydroxyvitamin D(3) (VD(3)) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.

Topics: Animals; Aquaporin 3; Biomarkers; Calcitriol; Calcium; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epidermal Cells; Epidermis; Homeostasis; Humans; Keratin-10; Keratin-5; Keratinocytes; Mice; Mice, Knockout; Protein Precursors; Tretinoin; Wound Healing

Sp1 is a transcription factor that regulates expression of mammalian and viral genes and is involved in different facets of cellular functions in eukaryotic cells. Here, we investigated Sp1 expression in primary mouse and human keratinocyte (KC) culture using quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy. Expression of Sp1 was post-transcriptionally up-regulated with increasing time in primary KC cultures. Sp1 expression, coincident with expression of human papillomavirus L1 capsid protein and involucrin, was associated with cell differentiation in vitro and in vivo in human and mouse skins. Immunoprecipitation experiments showed that Sp1 and L1 could be bound in a complex. Calcium (Ca(2+)) and all-trans retinoic acid are the positive modulators of KC differentiation, which positively and negatively regulated Sp1 and L1 expression. The data suggest that coincident expression of Sp1 with L1 proteins in differentiating KCs is mediated by a calcium-dependent signaling pathway.

Topics: Animals; Biomarkers; Blotting, Western; Calcium; Capsid Proteins; Cell Differentiation; Fluorescent Antibody Technique; Gene Expression; Humans; Keratinocytes; Mice; Oncogene Proteins, Viral; Protein Precursors; Sp1 Transcription Factor; Tretinoin; Up-Regulation; Viral Proteins

Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation.. Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay.. The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta.. The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.

Topics: Calcium; Calgranulin A; Calgranulin B; CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Cells, Cultured; Culture Media, Serum-Free; Filaggrin Proteins; Gene Expression Regulation; Gingiva; Humans; Interleukin-1alpha; Keratin-14; Keratinocytes; Leukocyte L1 Antigen Complex; Phosphoproteins; Protein Precursors; Transforming Growth Factor beta; Tretinoin

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

We studied the effect of all-trans retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun reached a maximum after 2 weeks in culture (1 week postconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simultaneous treatment with both retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation and stimulated cell differentiation resulting in acceleration of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly by inhibition of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were maintained. These results show that modulation of the composition of the medium by the addition of various vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation of the culture lifespan.

Topics: Alitretinoin; Calcitriol; Cell Differentiation; Cell Division; Cells, Cultured; Cornified Envelope Proline-Rich Proteins; Drug Evaluation, Preclinical; Drug Therapy, Combination; Humans; Keratinocytes; Membrane Proteins; Phenotype; Protein Precursors; Proteins; Proto-Oncogene Proteins c-jun; RNA, Messenger; Tretinoin

Involucrin is a major component of the cornified envelope and a marker for terminal differentiation in keratinocytes. We examined hormone regulation of involucrin mRNA expression and transcriptional modulation of the involucrin gene in human keratinocyte cell lines. Dexamethasone enhanced, and retinoic acid decreased, endogenous involucrin mRNA expression in cells treated with these ligands. Functional interactions between each ligand were observed; all-trans or 9-cis retinoic acid reduced dexamethasone enhancement of involucrin expression. Transcriptional control of the involucrin promoter reflected the responses observed for the endogenous gene. All-trans or 9-cis retinoic acid inhibited involucrin expression, and both retinoids antagonized glucocorticoid-mediated induction of reporter gene activity equally well. In contrast, a control promoter was unaffected by hormone treatment, indicating that stabilization of reporter gene mRNA or protein did not account for the observed differences in activity. These results demonstrate that transcriptional control plays a role in regulation of the endogenous involucrin gene by glucocorticoids and retinoids and that DNA sequences within the isolated promoter region confer hormone responsiveness. Hormone-mediated responses mapped to a proximal promoter region that contained a functional AP1 site. Stimulation of AP1 activity by phorbol ester was suppressed by retinoic acid, and cotransfection with a c-jun expression vector reversed the retinoic acid response. Based on these data, we suggest that suppression of involucrin promoter activity by retinoic acid may be mediated through interaction with the AP1 transcriptional complex.

Topics: Adaptor Protein Complex 1; Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Vesicular Transport; Antineoplastic Agents; Blotting, Northern; Cell Differentiation; Cell Line; Cell Membrane; Gene Expression Regulation; Glucocorticoids; Hormones; Humans; Keratinocytes; Membrane Proteins; Promoter Regions, Genetic; Protein Precursors; Receptors, Retinoic Acid; RNA, Messenger; Transcription, Genetic; Transfection; Tretinoin

Lysophosphatidic acid (LPA) is a biologically active phospholipid known to have growth factor-like activity on fibroblasts. Although the intracellular signal transduction pathways affected by LPA have been well characterized, the possibility that peptide growth factors are involved in the proliferative response of cells to LPA has not been thoroughly investigated. The focus of this work was to determine the effects of LPA on the proliferation and differentiation of early passage cultured human keratinocytes with emphasis on determining if transforming growth factors (TGF), types alpha and beta, are induced by LPA. The effects of LPA are compared with all-trans-retinoic acid (RA), a structurally unrelated lipid that has previously been shown to induce both TGF alpha and TGF beta and have pronounced effects on keratinocyte proliferation and differentiation. Treatment of cultured human keratinocytes with LPA or RA induced the production of TGF alpha by four- to eightfold. A number of structurally related phospholipids did not mimic the TGF alpha-inducing activity of LPA. LPA is mitogenic for keratinocytes and its stimulatory effect could be blocked with an inhibitory antibody to the EGF/TGF alpha receptor, suggesting that the induction of TGF alpha mediates LPA stimulation of keratinocyte proliferation. LPA and RA also induced both the active and latent forms TGF beta from cultured keratinocytes. Induction of TGF beta may mediate the effects LPA had on keratinocyte differentiation which were apparent by inhibition of proliferation (confluent cultures) and increased involucrin synthesis. Dramatic morphological changes were also observed after LPA treatment. Mechanistic studies suggest that LPA activates both pertussis toxin-sensitive and -insensitive signaling pathways involving protein kinase C activation and protein tyrosine phosphorylation. The effects of LPA on TGF alpha and TGF beta production by keratinocytes likely have in vivo relevance as concluded from rodent studies involving topical LPA treatments.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Epidermal Cells; Epidermis; ErbB Receptors; Female; Humans; Keratinocytes; Lysophospholipids; Mice; Mice, Hairless; Pertussis Toxin; Phosphatidic Acids; Protein Kinase C; Protein Precursors; Skin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tretinoin; Virulence Factors, Bordetella

Retinoic acid (RA) cream treatment alters epidermal proliferation and differentiation in photoaged skin.. To study the changes in the expression of markers of epidermal differentiation in photoaged skin following RA cream treatment.. Ten subjects with photoaged skin were examined before treatment and at regular intervals during 12 months of once daily application of 0.05% tretinoin cream over the left forearm. Paraffin-embedded biopsy sections from the forearm were stained with loricrin, filaggrin, involucrin and cornifin antisera in addition to hematoxylin and eosin. The various layers were measured using a calibrated optical micrometer.. The thickness of the epidermis increased rapidly, reaching a substantial increase after 1 month of retinoid cream application and retaining it during the following 12 months. The stratum granulosum achieved a transient but substantial increase after 1 and 3 months. Except involucrin, the ratio between the layers expressing the various markers of epidermal differentiation and the epidermis significantly increased following tretinoin cream treatment.. RA cream treatment not only increases the thickness of the epidermis but also the programming of late terminal epidermal differentiation, as expressed by the markers studied. Thus, RA appears to affect both proliferation and differentiation of keratinocytes in vivo.

Topics: Administration, Topical; Aged; Aged, 80 and over; Cell Differentiation; Cornified Envelope Proline-Rich Proteins; Epidermis; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratolytic Agents; Membrane Proteins; Protein Precursors; Skin Aging; Tretinoin

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

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

The expression of cornifin, a putative cross-linked envelope precursor, was investigated in several squamous differentiating tissues by in situ hybridization and immunohistochemical analysis. Cornifin mRNA and protein, which are absent in the normal mucociliary tracheal epithelium, are induced in the suprabasal layers of the squamous metaplastic tracheal epithelium of vitamin A-deficient hamsters. Similar to the induction of squamous metaplasia in vivo, culture of rabbit tracheal cells in the absence of retinoids results in squamous differentiation and expression of cornifin. This induction of cornifin expression is suppressed by retinoic acid and several of its analogs. Cornifin mRNA and protein are also detected in the suprabasal layers of the squamous epithelium of rabbit esophagus and tongue. The distribution of cornifin in human epidermis was compared with that of two other crosslinked envelope precursor proteins, involucrin and loricrin. The localization of cornifin and involucrin is very similar. Both are induced in the spinous layer and appear at an earlier stage during epidermal differentiation than loricrin. The expression of cornifin is greatly increased in psoriatic skin. Cornifin mRNA is barely detectable in normal epidermis, whereas it is present at relatively high levels in the suprabasal layers of psoriatic epidermis. Topical treatment with RA results in thickening of the skin and increases the level of cornifin mRNA and protein in the upper spinous layers of mouse skin. Cornifin expression correlates generally with squamous differentiation in a variety of tissues and is abnormally regulated in psoriatic skin and in skin treated topically with retinoic acid.

Topics: Administration, Topical; Animals; Cells, Cultured; Cornified Envelope Proline-Rich Proteins; Cricetinae; Esophagus; Gene Expression; Humans; Male; Membrane Proteins; Mice; Protein Precursors; Psoriasis; Rabbits; RNA, Messenger; Skin; Trachea; Tracheal Neoplasms; Tretinoin; Vitamin A Deficiency

Double histochemical staining followed by flow cytometric analysis was performed to determine whether involucrin synthesis is associated with a particular phase of the cell cycle. In low-calcium medium (0.1 mM) monolayer cultures the expression of involucrin was confined to about 8% of the total cell populations. When the concentration of calcium was increased to 1.8 mM, the percentage increased and the distribution pattern of the cell cycle changed. The addition of retinoic acid at concentrations in the range 10(-8) M to 10(-6) M to a high-calcium medium induced a further increase. The greatest increase in involucrin expression (up to five-fold) occurred in both the G1/G0 and G2 + M phases, while S phase cells showed a two-fold increase. The results indicate that involucrin synthesis is induced by retinoic acid, which occurs at any stage of the cell cycle, even in the S phase.

Topics: Calcium; Cell Cycle; Cell Differentiation; Cells, Cultured; Flow Cytometry; Humans; Keratinocytes; Protein Precursors; Tretinoin

Inducing the expression of ICAM-1 (CD54) on the surface of epidermal keratinocytes is an important step in initiating leukocyte interaction with the epidermis. We studied the effect of keratinocyte differentiation and of drugs used to treat epidermal inflammation on the induction of this important adhesion molecule. Cell membrane expression of ICAM-1 in cultured human keratinocytes was analyzed using both immunofluorescence and FACS analysis of staining with anti-ICAM-1 monoclonal antibody and was correlated with markers of keratinocyte differentiation. Cell-surface ICAM-1 expression was induced by gamma interferon in all culture conditions, but was significantly greater (p less than 0.014) in cells grown in low-calcium medium ([Ca++] 0.03 mM), and correlated with increased staining for the basal cell keratin K5. The synthetic retinoid Etretin (Ro 10-1670) enhanced the interferon-induced ICAM-1 expression over a wide concentration range (10(-8)-10(-5) M); however, this effect was only seen in the more differentiated cells grown in 0.15 mM and 1.0 mM calcium and not in the cells grown in 0.03 mM calcium. The Etretin effects on intracellular K5 staining paralleled those on cell-surface ICAM-1. Anti-inflammatory glucocorticoids had no effect on ICAM-1 expression in cultured human keratinocytes, even at suboptimal gamma interferon doses (5 U/ml). beta-estradiol, on the other hand, mimicked the Etretin effect, increasing both IFN induction of ICAM-1 expression and K5 staining in more differentiated keratinocytes in 0.15 and 1.0 mM calcium, but not in those in 0.03 mM calcium. Both Etretin and beta-estradiol decreased staining of involucrin, a marker of terminal differentiation, supporting the proposition that in this experimental system these drugs suppress keratinocyte differentiation. The enhanced ICAM-1 induction in keratinocytes with a basal level of differentiation correlates with the in vivo effects of interferon on ICAM-1 and may be a principal determinant in the patterns of ICAM-1 seen in inflammatory skin diseases.

Topics: Acitretin; Cell Adhesion Molecules; Cell Differentiation; Estradiol; Glucocorticoids; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Keratinocytes; Male; Protein Precursors; Staining and Labeling; Tretinoin

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

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

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

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

A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10(-3) M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells when compared to normal human keratinocytes. In other respects, EGF, which inhibits the proliferation of A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Membrane; Cells, Cultured; Culture Media; Epidermal Growth Factor; Epidermis; Female; Humans; Protein Precursors; Transglutaminases; Tretinoin

A malignant human cell line (SqCC/Y1) derived from a squamous carcinoma of the buccal mucosa is described. It formed a stratified cellular structure with ultrastructural characteristics of a fully differentiated stratified squamous epithelium when cultured in equal parts of Dulbecco's modified Eagle medium and Ham's medium F12, supplemented only with insulin, transferrin, and selenium. After 14 days in culture in this defined medium, 30% of the cells became keratinized (insoluble in detergent), and 75% of the cells were capable of being induced to form cornified cell envelopes. Involucrin, the precursor protein of the cornified cell envelope, could be detected by immunofluorescence only in suprabasal cells. Treatment of SqCC/Y1 cultures with 5 X 10(-8) M all-trans-retinoic acid (RA) completely inhibited stratification and markedly increased cell desquamation. In the presence of RA, less than 10% of the cells became keratinized, and only 15-20% of the cells acquired envelope-forming competence. The fraction of colony-forming cells in RA-treated cultures was tenfold higher than in fully mature cultures. Thus RA appears to be an effective inhibitor of terminal differentiation of SqCC/Y1 cells.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Culture Media; Humans; Neoplastic Stem Cells; Protein Precursors; Tretinoin