epidermal-growth-factor and involucrin

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.

Topics: Blotting, Western; Calcium Chloride; Cell Differentiation; Cells, Cultured; Epidermal Growth Factor; Epidermis; Fluorescent Antibody Technique; Humans; Immunophenotyping; Kallikreins; Keratin-10; Keratin-14; Keratin-5; Keratinocytes; Mesenchymal Stem Cells; Microscopy; Protein Precursors

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered--inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca2+ content formed monolayers, which after elevation of Ca2+ produced multilayers with cells containing keratohyalin-like granules. Ca2+ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca2+ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.

Topics: Animals; Aspartic Acid Endopeptidases; Calcium; Cell Differentiation; Cells, Cultured; Epidermal Growth Factor; Female; Filaggrin Proteins; Gene Expression Regulation; Hair Follicle; Intermediate Filament Proteins; Keratinocytes; Keratins, Hair-Specific; Microscopy, Electron, Transmission; Organ Specificity; Paracrine Communication; Protein Precursors; Rats; Rats, Wistar

Epidermal homeostasis and repair of the skin barrier require that epidermal keratinocytes respond to alterations of their environment. We report that cellular stress with methyl-beta-cyclodextrin (MBCD), a molecule that extracts membrane cholesterol and thereby disrupts the structure of lipid rafts, strongly induces the synthesis of heparin-binding EGF-like growth factor (HB-EGF) in keratinocytes through the activation of p38 mitogen-activated protein kinase. Interesting parallels between lipid raft disruption and oxidative stress can be drawn as hydrogen peroxide induces p38 activation and HB-EGF synthesis in keratinocytes. Consistent with other studies, we show increased HB-EGF expression in keratinocytes located at the margin of wounded skin areas. Analyzing cultured keratinocytes exposed to rhHB-EGF, we report increased HB-EGF mRNA levels and alterations in the expression of differentiation markers. Interestingly, identical alterations in differentiation markers are shown to occur in vivo at the wound margin and in HB-EGF-treated cultures. In addition, in vitro sectioning of skin samples also induces the expression of HB-EGF at the border of the incisions. Altogether, our data suggest that expression of HB-EGF is a marker of the keratinocyte's response to a challenging environment and demonstrate that this growth factor alters the phenotype of keratinocytes in a manner similar to that observed during epidermal repair.

Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Cholesterol; Epidermal Cells; Epidermal Growth Factor; Epidermis; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Keratin-10; Keratinocytes; Membrane Microdomains; Organ Culture Techniques; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Protein Precursors; Up-Regulation; Wound Healing

A suitable model analyzing the behavior of well-differentiated squamous cell carcinoma has not yet been established. We tried to establish such a system using a reconstructed oral mucosa, in which T3M-1 squamous cell carcinoma cells were cultured on 3T3 fibroblast-containing collagen gel. Fibroblasts promoted the stratification and keratinization of T3M-1 cells. During growth, the Ki-67 index of T3M-1 cells with fibroblasts was higher than that of T3M-1 cells alone. Fibroblasts increased the expression of involucrin, a differentiating marker of keratinocytes, in T3M-1 cells. They also promoted the invasion of T3M-1 cells into the gel. When T3M-1 cells alone were cultured in a fibroblast-conditioned (FC) medium, the fibroblast-induced phenomena mentioned above were almost replicated. In addition, epidermal growth factqr (EGF) promoted T3M-1 cells growth, but not the invasion. cDNA microarray analysis showed that FC medium increased the expression of EGF receptor and several other mRNAs of T3M-1 cells. The data suggest that T3M-1 cells, under cancer-stromal fibroblast interaction, undergo invasive growth with their well-differentiated squamous phenotype, and that this interaction may be mediated partly by soluble molecules (e.g., EGF) in an autocrine or paracrine pathway. Our system will probably provide a useful model for analyzing the biological behavior of well-differentiated squamous cell carcinoma.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Coculture Techniques; Collagen; Culture Media, Conditioned; Epidermal Growth Factor; Fibroblasts; Gels; Hepatocyte Growth Factor; Humans; Ki-67 Antigen; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Protein Precursors; Receptors, Interleukin-1; Stromal Cells

We have previously demonstrated that phospholipase C (PLC)-gamma1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-gamma1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-gamma1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-gamma1 by epidermal growth factor, calcium-induced activation of PLC-gamma1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3 binds to and activates PLC-gamma1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85alpha, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-gamma1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-gamma1 via increased PIP3 formation mediated by c-src- and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.

Topics: Blotting, Western; Calcium; Cell Differentiation; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Genes, Dominant; Humans; Immunoprecipitation; Keratinocytes; Models, Biological; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphorylation; Protein Precursors; src-Family Kinases; Time Factors; Transfection; Transglutaminases; Tyrosine

Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal-epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.

Topics: Animals; Cell Transplantation; Combined Modality Therapy; Epidermal Cells; Epidermal Growth Factor; Epidermis; Fibrin Tissue Adhesive; Humans; Immunohistochemistry; Keratinocytes; Laminin; Male; Mice; Mice, Nude; Protein Precursors; Regeneration; Skin; Wound Healing

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.

Topics: Actins; beta Catenin; Cell Differentiation; Cell Division; Cells, Cultured; Cytoskeletal Proteins; Dose-Response Relationship, Radiation; Electricity; Electromagnetic Fields; Epidermal Growth Factor; Humans; Keratinocytes; Mouth; Protein Precursors; Radiation Dosage; Tissue Distribution; Trans-Activators

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

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Differentiation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Male; Protein Precursors; RNA, Messenger; Transglutaminases; Tumor Cells, Cultured

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

The A431 cell line is composed of malignant keratinocytes derived from a vulval epidermoïd carcinoma. These cells have the peculiarity to stop their proliferation when they are treated with physiological concentrations of EGF, which is a mitogen for normal keratinocytes. We reported earlier that EGF induces involucrin accumulation in A431 cells and proposed that the arrest of proliferation triggers differentiation as shown by the induction of this cornified envelope precursor protein. To test this hypothesis, we compared the A431 subclone 15, which is not growth arrested by EGF-treatment, to the parental A431 cells. We found indeed that EGF reduces the involucrin content of clone 15 cells in a dose dependent manner. These opposite effects of EGF on the expression of terminal differentiation marker involucrin in A431 and A431 clone 15 keratinocytes were observed in defined medium as well as in presence of fetal calf serum. Nevertheless, when growth of parental A431 cells was inhibited by treatment with TGF-beta or simply when cultures reached confluency, no involucrin accumulation was observed. Therefore growth arrest per se is not directly correlated with the induction of differentiation.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Division; Culture Media; Epidermal Cells; Epidermal Growth Factor; Humans; Mice; Protein Precursors; Transforming Growth Factors; Tumor Cells, Cultured

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

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

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