Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders.

Background: Retinoid-based therapies are commonly used in the treatment of disorders of keratinization and other skin disorders but can result in non-specific effects and adverse reactions. Use of retinoic acid metabolism blocking agents (RAMBAs) such as DX308 may address these shortcomings. Objectives: Characterize the therapeutic potential of recently discovered, CYP26-selective RAMBA, DX308. Materials and Methods: Preliminary in vitro assessment of potential off-target activity, metabolic and toxicologic profiling. Studies to assess safety and efficacy of topical treatment in correcting abnormal skin morphology in rhino mice. Extensive gene expression profiling by RNA sequencing and qPCR in 3D epidermis grown with keratinocytes (KCs) from keratinization disorders and healthy controls, to investigate modulation of retinoid biopathways. Results: In vitro, DX308 does not interact with off-target nuclear receptors or CYP450s, is not genotoxic, and is stable in skin, despite vigorous hepatic metabolism. In vivo, topical DX308 induces comedolysis and epidermal thickening without apparent adverse effects. Gene expression profiling shows potent modulation of retinoid-responsive genes by DX308 in both healthy and keratinization disorder KCs. Pathway analysis suggests DX308 may inhibit inflammatory and immune responses in KCs. Conclusions: These preliminary studies suggest that DX308 is an efficacious topical therapeutic with a favourable metabolic and safety profiles. DX308 may present an improved therapeutic alternative for the treatment of keratinization disorders and other retinoid-responsive skin ailments.

The CYP26 inhibitor R115866 potentiates the effects of all-trans retinoic acid on cultured human epidermal keratinocytes.

BACKGROUND: 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. OBJECTIVES: To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes. METHODS: 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. RESULTS: 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. CONCLUSION: These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

Immunogold silver staining associated with epi-fluorescence for cucumber mosaic virus localisation on semi-thin sections of banana tissues.

The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone.

Modelling atopic dermatitis during the morphogenetic process involved in reconstruction of a human epidermis.

Most crucial role of epidermis is to maintain efficient barrier between the organism and its environment. This barrier is however perturbed in inflammatory skin conditions like atopic dermatitis (AD), one common chronic disease. This review depicts characteristics of a model intending to reproduce epidermal features of AD in vitro. Firstly, methyl-ß-cyclodextrin (MßCD) during reconstruction of epidermis was used to deplete cholesterol from plasma membrane because this condition reproduces characteristics of AD at transcriptomic level in monolayer cultures. Major changes are confirmed after same treatment inside reconstructed human epidermis (RHE). However, since early treatment do not reveal impairment to reconstruct a functional epidermal barrier and given the importance of the Th2 dysregulated immune response in AD, cholesterol-depleted RHE at day 11 of reconstruction were then incubated with three Th2-related cytokines (IL-4, IL-13 and IL-25) previously reported as playing important roles in the development of AD, as well as altering overall function of epidermal barrier. When combining both treatments, essential epidermal features of AD are observed. Indeed, RHE then exhibit spongiosis, disappearing granular layer, alteration of barrier function, as well as dysregulated expression levels for genes involved in AD pathogenesis. Moreover, while trying to identify individual roles for each component used to create AD-like alterations, incubation with IL-4 following cholesterol depletion from plasma membrane was found inducing most of the reported alterations. This model suggests potential for better investigations of epidermal AD features and may be considered for eventual in vitro screening of cosmetics or therapeutic compounds.

Preparation and characterizations of EGDE crosslinked chitosan electrospun membranes.

Composite Crosslinked nanofibrous membranes of chitosan, ethylene glycol diglycidyl ether (EGDE) and polyethylene oxide was successfully prepared with bead free morphology via electrospinning technique followed by heat mediated chemical crosslinking. Architectural stability of nanofiber mat in aqueous medium was achieved by chemical crosslinking of only 1% EGDE, and tensile strength tests revealed that increasing EGDE content has considerably enhance the elastic modulus of nanofibers. The structure, morphology and mechanical properties of nanofibers were characterized by Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM) and Instron machine, respectively. Skin fibroblasts and endothelial cells showed good attachment, proliferation and viability on crosslinked electrospun membranes. The results indicate a good biocompatibility and non-toxic nature of the resulted membrane.

Specific internalization of basal membrane domains containing the integrin alpha 6 beta 4 in dispase-detached cultured human keratinocytes.

The integrin alpha 6 beta 4 is polarized towards the basal side of basal keratinocytes and helps anchor these cells to the basement membrane components. We have found that cultured human epidermal keratinocytes, when detached from their culture substratum, as for grafting, using the enzyme dispase, rapidly internalize the basal membrane domains containing the integrin alpha 6 beta 4, while integrins of the very late antigen subtype remain on the cell surface. Detachment and incubation at 4 degrees C prevent this internalization, as well as the contraction of the detached sheet area. Subsequent incubation at 37 degrees C initializes this contraction and allows the basal integrin alpha 6 beta 4 to be internalized. We took advantage of this blockage to label upon detachment using immunogold techniques, the alpha 6 subunit present on the basal cell surface; then we studied its internalization with the electron microscope. This internalization pathway differs from classical receptor-mediated endocytosis, and intermediate filaments might possibly play a role in this process. Interestingly, 1 h after their internalization from the basal membrane, a third of the gold particles labeling the alpha 6 subunit was found between lateral membranes of basal cells, strongly suggesting that the integrin alpha 6 beta 4 can be partly recycled to the cell surface in these conditions.

The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility.

Integrins are involved in several ways in keratinocyte physiology, including cell motility. CD9 is a member of the tetraspanin protein family which is found in association with other transmembrane proteins like the integrins. CD9 is expressed in the epidermal tissue, but this expression is not regulated by differentiation. The present work focuses on association of CD9 with the integrin alpha6beta4 in keratinocytes. In vivo, CD9 does not co-localize with alpha6beta4, and is not internalized with the integrin upon basal detachment with dispase. In vitro, CD9 is found partly in co-localization with alpha6beta4 and is internalized with the integrin after keratinocyte detachment with dispase. Using blocking antibodies in a phagokinetic tracks assay, we show that CD9, and to a lesser extent alpha6beta4, but not the tetraspanin CD82, promote motility of subconfluent keratinocytes on collagen I. Our observations also suggest that CD9 is involved in the formation of lamellipodia. We also report that the phorbol ester TPA has no effect on CD9 expression and association with alpha6beta4, but increases keratinocyte motility, possibly through modulation of integrin subunits expression, or through upregulation of collagenase-1 expression. Together, these results confirm that CD9 associates with alpha6beta4 in cultured keratinocytes, possibly in order to regulate the function of the integrin, and that CD9 is involved in keratinocyte motility on collagen. The data suggest that regulation of adhesion characteristics by CD9 in keratinocytes may play a role in epidermal repair.

Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal K1/K10 keratins.

Irreversible growth arrest and commitment to differentiation are among the earliest events in the program of cellular terminal differentiation. The transition from highly proliferative human keratinocytes in subconfluent culture to stationary cells in confluent culture was studied in a serum-free culture system to identify conditions that regulate the initiation of terminal differentiation in keratinocytes. We observed that culture confluence strongly induced commitment to terminal differentiation, as demonstrated by a dramatic loss of keratinocyte clonogenicity. Commitment was accompanied by the rapid induction of early differentiation markers, represented by expression of suprabasal keratin 1 (K1) and 10 (K10) genes. Induction of differentiation was independent of low (0.1 mM) or high (1.5 mM) calcium concentration in the medium. Epidermal growth factor suppressed expression of K1 and K10 mRNA in cultures induced to differentiate. Suspension of keratinocytes in methylcellulose medium failed to induce in subconfluent cultures, or enhance in confluent cultures, the expression of K1 and K10 genes. Subconfluent cells cultured in medium containing high calcium and no exogenous growth factor induced expression of K1 and K10 transcripts, but commitment and loss of proliferative potential were not observed. Confluent cell density primarily controlled keratinocyte commitment to terminal differentiation and differentiated keratin gene expression. However, suprabasal K1 and K10 gene expression also was regulated by medium calcium and exogenous growth-factor concentrations in subconfluent cultures that promoted cell-cell association. Epidermal growth factor inhibited the expression of suprabasal keratins but not the commitment to terminal differentiation mediated by cell confluence. Control of keratinocyte commitment and expression of selected differentiation genes are mediated by cell confluence and, at subconfluence, by specific culture factors.

Basal detachment of the epidermis using dispase: tissue spatial organization and fate of integrin alpha 6 beta 4 and hemidesmosomes.

Dispase has been utilized to produce basal detachment of the epidermis of human skin biopsies and to study the consequences induced afterwards during incubations of the detached tissue. Spatial reorganization of the epidermis is observed under these conditions and is characterized by disappearance of the typical basal keratinocyte layer. Immunofluorescent labelings reveal upward migration of several cells exhibiting the basal phenotype between suprabasal differentiating keratinocytes and demonstrate progressive intracellular expression of hemidesmosomal components: the integrin alpha 6 beta 4 and two plaque components, the 230-kDa bullous pemphigoid antigen and HD1, a 500-kDa protein. Using electron microscopy and immunogold techniques, we demonstrate that the hemidesmosome-containing basal membrane domains enter the cell cytoplasm after detachment of the epidermal tissue. Partial recycling of internalized hemidesmosomal components is also suggested. Our findings illustrate the processing of released hemidesmosomes in detached basal keratinocytes and suggest some heterogeneity between basal cells migrating towards a suprabasal position and those remaining in the basal layer. These results suggest that the dispase-detached epidermis is a self-remodeling tissue in which basal keratinocytes' and tissue's polarities observed in the anchored epidermis are progressively changing.

Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes.

The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.

Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) inhibitor KN-62 suppresses the activity of mitogen-activated protein kinase (MAPK), c-myc activation and human keratinocyte proliferation.

Autocrine growth of human epidermal keratinocytes can be maintained in subconfluent cell cultures in the absence of exogenous growth factors. We used this culture model to investigate the interactions between the mitogen-activated protein kinase (MAPK) pathway and Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) in autocrine keratinocyte proliferation. We have previously demonstrated that MAPK and protein kinase C (PKC) are both involved in keratinocyte proliferation in a complex set of interactions. Treatment of keratinocytes with PD98059, a potent inhibitor of MAPK kinase, inhibited the MAPK pathway, c-myc activation and autocrine keratinocyte proliferation. Application of the CaM-kinase inhibitor KN-62 also led to a strong inhibition of MAPK/c-myc activation and autocrine keratinocyte proliferation. Other inhibitors, such as wortmannin (selective and potent inhibitor of phosphatidylinositol 3-kinase) and AG 490 (JAK2 inhibitor) had weak effects on autocrine keratinocyte proliferation, MAPK and c-myc activation. Our results clearly demonstrate a crosstalk between CaM-kinase/MAPK pathways in transducing keratinocyte proliferation stimuli.

A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies.

The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 m M) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1alpha and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.

Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: a potential pathway for delivering therapeutic drugs and genes.

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4 degrees C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 micrograms/ml. ApoE-free HDL3 or acetylated LDL did not significantly compete with 125I-LDL binding, up to 500 micrograms/ml. 125I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 micrograms/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37 degrees C, 125I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.

Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals.

In the present study, a model of reconstructed human epidermis (RHE) was used as an in vitro model to discriminate the effects of Tween 80, Triton X100 and benzalkonium chloride (BC) as irritants and 1-chloro-2,4-dinitrobenzene (DNCB) as sensitizing agent. The extent of epidermal irritation and sensitization was evaluated morphologically and on the basis of intracellular and extracellular levels of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8). The corresponding constitutive mRNA levels were quantified and the cytotoxic response was assessed by MTT assay. The RHE resembled normal human epidermis with all typical layers. The keratin 10 (K10) was typically confined in the suprabasal layers of the tissue, suggesting normal epidermal terminal differentiation. Topical application of the three surfactants resulted in significant changes of tissue morphology and was coupled with different dose-dependent decreases in cell viability corresponding to their in vivo irritant potency. The IL-1alpha release increased concomitantly with cell viability decrease, but surprisingly, the surfactants did not elicit elevated IL-8 levels. In contrast, DNCB did not induce elevated IL-1alpha release although it induced a rapid dose-dependent decrease in cell viability. On the contrary, it increased the IL-8 release. RT-PCR demonstrated the presence of mRNA for IL-1alpha as well as for IL-8. IL-1alpha was the most abundant cytokine transcript. BC, Triton X100 and DNCB upregulated IL-8 mRNA expression while only BC induced a significant increase in IL-1alpha mRNA expression. Our results showed that the production of IL-1alpha and its release into the extracellular medium was ascribed not only to direct cytotoxicity but also to the extent of tissue stimulation. Conversely, the production of IL-8 did not directly correlate with cytotoxicity but seems to be linked to the type of product applied either irritant or sensitizer. Our data demonstrate that divergent IL-1alpha and IL-8 release profiles and corresponding mRNA upregulation characterize the response to the tested irritants and DNCB. These results currently need confirmation by the introduction of more numerous known irritants and sensitizers in the tests used in the present study. However, they already suggest that it may be possible in a single assay to classify and to discriminate between irritant and sensitizing agents as a function of induced cytokine production patterns and of cell viability measurements.

Analysis of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization.

In the present study, reconstructed human epidermis (RHE) was used as an in vitro model to discriminate 1-chloro-2,4-dinitrobenzene (DNCB), nickel sulfate (NiSO(4)), oxazolone (OXA), 2,4-dinitrofluorobenzene (DNFB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as skin sensitizers from benzalkonium chloride (BC), benzoic acid (BA) and sodium lauryl sulfate (SLS) as skin irritants. Our criteria were (a) the differential IL-1alpha and IL-8 synthesis and release (b) cytotoxicity assessment by MTT assay. When the RHE are topically treated with the sensitizers, very low levels of extra- and intracellular IL-1alpha are observed although they induce significant cytotoxicity. In contrast, they exhibit a sharp maximum of IL-8 release. In the presence of the tested irritants, we observe the inverse cytokine release profile, although they induce dose-dependent cytotoxicity profiles similar to those observed with the sensitizers. Finally, IL-1alpha mRNA upregulation is observed after topical application of both sensitizers and irritants, but only the latter significantly increase extracellular IL-1alpha. In conclusion, our results suggest that the associated determination of IL-8, with IL-1alpha, and MTT conversion are at least necessary to discriminate and classify, in a single assay, irritant and sensitizing agents and represent a potential in vitro alternative to two classical in vivo assays.

Utilization of human cultured epidermal keratinocytes: irreversibility of the inhibition of proliferation induced in stored detached cultures.

In order to look for the best conditions required for the utilization of cultured epithelium in the treatment of burn wounds, some experiments have been performed studying the storage of dispase-detached cultures. The viability of keratinocytes and, after trypsin dissociation of the cultures, the adhesion and proliferative potential of living keratinocytes were measured. Our laboratory investigations suggest that the storage period of detached cultures has to be kept as short as possible to preserve the keratinocytes' growth potential.

Endocytosis of low density lipoproteins in human endothelial cells: typical morphological aspects of the high affinity receptor-mediated pathway as revealed by serial sections and acid phosphatase cytochemistry.

We used electron microscopy serial sections and acid phosphatase cytochemistry to follow the endocytosis of low density lipoproteins (LDL) by cultured human umbilical vein endothelial cells. The surface LDL-receptors were labeled with LDL-colloidal gold conjugates at 4 degrees C and then, cells were incubated for 0, 5, 10, 20, 30 and 60 min at 37 degrees C before fixation. The organelles identified in this way and studied by serial sections corresponded to those described in endothelial cells and in other cell types, with a peripheral endosomal compartment and a juxtanuclear endosomal compartment, located in the vicinity of the Golgi apparatus and mainly composed of round vesicle-containing structures called multivesicular bodies. By acid phosphatase cytochemistry, the limits between the endosomal juxtanuclear compartment and the lysosomal compartment were also studied and it was shown that multivesicular bodies are probably the first site for the acquisition of acid hydrolases. Lastly, the observation of highly gold-labeled endothelial cells when cells were incubated for 90 min at 37 degrees C with the LDL-gold conjugate, suggested that LDL was degraded in the lysosomal compartment.

Influence of a short oxidative stress on the LDL endocytosis by human endothelial cells: an ultrastructural study.

Following injury induced by oxidant stress (exposure to xanthine-xanthine oxidase during 120 min), cultured human umbilical vein endothelial cells were severely modified. These lesions were evaluated by electron microscopy and the types of injury were shown to be progressive cell blebbing as well as altered mitochondrial features. Enlargement of the endoplasmic reticulum and swelling of the Golgi apparatus were also observed. With the intention of learning more about the role of oxygen-derivative-induced alterations of the endocytotic apparatus (plasma membrane, endosomes, lysosomes), the receptor-mediated endocytosis of colloidal gold-conjugated LDL was evaluated. It was demonstrated that this endocytosis is drastically decreased following this type of injury, corroborating our previous report of important reduction in 125I-LDL endocytosis under the same conditions. Tubular electron-lucent structures, often observed in the vicinity of blebs, could possibly be involved in the impairment of LDL receptor accessibility.

In Vitro models of epidermal differentiation.

The differentiation program followed by the epidermal keratinocyte in skin is intended to continuously produce and maintain a cornified layer made of fully keratinized cells. This outer layer of the body provides a certain protection against external pathogens and chemical or physical agents, together with a barrier that prevents loss of body fluids. Considerable knowledge of epidermal differentiation and understanding of its regulation has progressively emerged from the availability of keratinocyte cultures, and from the consecutive possibility of unlimited in vitro experimentation. This short review briefly presents the main current in vitro models of epidermal differentiation and emphasises their advantages of pitfalls when studying particular steps of the differentiation program or analyzing their regulation.