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The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate-whether to remain proliferative or terminally differentiate-is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome-wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non-differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock-down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that-in our in vitro experimental setup-it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation., (© 2020 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Background: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing., Objective: We aimed to investigate the effects of human β-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration., Methods: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation., Results: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction., Conclusion: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining., (© 2017 S. Karger AG, Basel.)

The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of dynamically regulated genes and p63-bound regulatory elements that are relevant for epithelial development and related diseases. p63-bound regulatory elements occur as single or clustered enhancers, and remarkably, only a subset is active as defined by the co-presence of the active enhancer mark histone modification H3K27ac in epidermal keratinocytes. We show that the dynamics of gene expression correlates with the activity of p63-bound enhancers rather than with p63 binding itself. The activity of p63-bound enhancers is likely determined by other transcription factors that cooperate with p63. Our data show that inactive p63-bound enhancers in epidermal keratinocytes may be active during the development of other epithelial-related structures such as limbs and suggest that p63 bookmarks genomic loci during the commitment of the epithelial lineage and regulates genes through temporal- and spatial-specific active enhancers., (© 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression, and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.

The interaction between keratinocytes and immune cells plays a major role in the development of inflammatory skin diseases like psoriasis and atopic dermatitis. Pharmacological intervention to inhibit T cell-derived proinflammatory mediators is an effective therapy in the treatment of psoriasis. Here, we present a model to study the interaction between keratinocytes and T cells in a three-dimensional (3D) microenvironment, based on human skin equivalents populated with CD4+ T cells. T cell migration into the dermis initiated keratinocyte activation within 2 days, with hallmarks of a psoriasiform inflammation after 4 days. Expression of epidermal psoriasis marker genes was upregulated, and proinflammatory cytokines and chemokines were highly expressed. Disturbed epidermal differentiation was shown by downregulated filaggrin expression and involucrin expression in the spinous layer. These effects were mediated via soluble factors produced by the T cells. The psoriasiform inflammation was also observed using T helper type 1 (Th1)- and Th17-polarized CD4+ T cells. We validated our model by treatment with anti-inflammatory drugs that reduced the expression of proinflammatory cytokines and chemokines and suppressed the psoriasiform inflammation. We propose that our T cell-driven inflammatory skin equivalent model has potential to study the pathogenesis of inflammatory skin diseases and may serve as a preclinical screening tool for anti-inflammatory drugs.

Reepithelialization of skin wounds is essential to restore barrier function and prevent infection. This process requires coordination of keratinocyte proliferation, migration, and differentiation, which may be impeded by various extrinsic and host-dependent factors. Deep, full-thickness wounds, e.g., burns, are often grafted with dermal matrices before transplantation of split-skin grafts. These dermal matrices need to be integrated in the host skin and serve as a substrate for neoepidermis formation. Systematic preclinical analysis of keratinocyte migration on established and experimental matrices has been hampered by the lack of suitable in vitro model systems. Here, we developed an in vitro full-thickness wound healing model in tissue-engineered human skin that allowed analysis of the reepithelialization process across different grafted dermal substitutes. We observed strong differences between porous and nonporous matrices, the latter being superior for reepithelialization. This finding was corroborated in rodent wound healing models. The model was optimized using lentivirus-transduced keratinocytes expressing enhanced green fluorescent protein and by the addition of human blood, which accelerated keratinocyte migration underneath the clot. Our model shows great potential for preclinical evaluation of tissue-engineered dermal substitutes in a medium-throughput format, thereby obviating the use of large numbers of experimental animals., (© 2013 by the Wound Healing Society.)

Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics.

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

Background: Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response., Objective: We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production., Methods: A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry., Results: Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells., Conclusion: We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases., (Copyright © 2012. Published by Elsevier Ireland Ltd.)

The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.

Background: Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed., Objectives: To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins., Methods: We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls., Results: Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD., Conclusions: Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)

A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar-structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Melanoma progression is a multistep progression from a common melanocytic nevus through the radial growth phase, the invasive vertical growth phase finally leading to metastatic spread into distant organs. Migration and invasion of tumor cells requires secretion of proteases to facilitate remodeling of the extracellular matrix including basement membranes. Here we used a reconstructed skin model to investigate melanoma growth and invasion in vitro. Using this model we show that the dermoepidermal basement membrane prevents the invasion of metastatic melanoma BLM and MV3 cells in the absence of a stratified epidermis. In the reconstructed skin model, matrix metalloproteinase-9, a protease activated early in melanoma development, is secreted by the keratinocytes and subsequently activated by an unknown soluble factor secreted by the melanoma cells. The dynamic interplay between keratinocytes and melanoma cells is further shown by an altered growth pattern of melanoma cells and the finding that a reconstructed epidermis induces invasion. Overall, our findings show that the invasive behavior of melanoma cells is determined by the melanoma cells themselves, but that the interplay between surrounding keratinocytes and the melanoma cells plays an important role in melanoma invasion.

Background: Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis (AD) have revealed genes with disease-specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease-specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general., Objectives: To analyse if gene expression signatures as found in purified epidermal cells from AD are also present in other eczematous conditions such as allergic and irritant contact dermatitis., Methods: We used real-time quantitative polymerase chain reaction, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analysed for comparison., Results: Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in AD epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, AD and allergic contact dermatitis, but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response., Conclusions: These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.

Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-alpha, interleukin (IL)-1alpha, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-alpha was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.

Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression.

Background: Epithelia are barrier-forming tissues that protect the organism against external noxious stimuli. Despite the similarity in function of epithelia, only few common protective mechanisms that are employed by these tissues have been systematically studied. Comparative analysis of genome-wide expression profiles generated by means of Serial Analysis of Gene Expression (SAGE) is a powerful approach to yield further insight into epithelial host defense mechanisms. We performed an extensive comparative analysis of previously published SAGE data sets of two types of epithelial cells, namely bronchial epithelial cells and keratinocytes, in which the response to pro-inflammatory cytokines was assessed. These data sets were used to elucidate a common denominator in epithelial host defense., Results: Bronchial epithelial cells and keratinocytes were found to have a high degree of overlap in gene expression. Using an in silico approach, an epithelial-specific molecular signature of gene expression was identified in bronchial epithelial cells and keratinocytes comprising of family members of keratins, small proline-rich proteins and proteinase inhibitors. Whereas some of the identified genes were known to be involved in inflammation, the majority of the signature represented genes that were previously not associated with host defense. Using polymerase chain reaction, presence of expression of selected tissue-specific genes was validated., Conclusion: Our comparative analysis of gene transcription reveals that bronchial epithelial cells and keratinocytes both express a subset of genes that is likely to be essential in epithelial barrier formation in these cell types. The expression of these genes is specific for bronchial epithelial cells and keratinocytes and is not seen in non-epithelial cells. We show that bronchial epithelial cells, similar to keratinocytes, express components that are able to form a cross-linked protein envelope that may contribute to an effective barrier against noxious stimuli and pathogens.

Recently, it was shown that lesional skin of atopic dermatitis patients expresses low levels of some antimicrobial peptides, compared with psoriasis patients. Here we performed microarray analysis on mRNA from purified lesional epidermal cells of patients with chronic plaque psoriasis and chronic atopic dermatitis, to investigate whether this is a general phenomenon for host defense proteins, and how specific it is for this class of molecules. Microarray data were confirmed on a selected set of genes by quantitative PCR and at the protein level by immunohistochemistry. We found overexpression of many antimicrobial proteins in keratinocytes from psoriatic skin compared with atopic dermatitis skin. Interestingly, we observed that markers of normal differentiation and the activated/hyperproliferative epidermal phenotype were expressed at equal levels. Chronic lesions of psoriasis and atopic dermatitis patients are remarkably similar with respect to cellular proliferation. We conclude that psoriatic epidermis expresses high levels of host defense proteins compared with atopic dermatitis epidermis, and this phenomenon appears to be specific for these proteins. It remains to be investigated whether this is caused by genetic polymorphisms in pathways leading to an epidermal antimicrobial response, or by differences in the cellular infiltrate in psoriasis compared with atopic dermatitis.

A model that explains how maintenance of normal homeostasis in human epidermis is achieved describes a heterogeneous cell population of stem cells (SC) and transit amplifying cells (TAC). There must be a tightly regulated balance between SC self-renewal and the generation of TAC that undergo a limited number of divisions before giving rise to postmitotic, terminally differentiated cells. To investigate whether this balance is disturbed in psoriatic epidermis, we have characterized flow sorted enriched SC and TAC using immunocytochemistry, flow cytometry, and real-time quantitative PCR. Our data demonstrate phenotypical and functional differences in SC (beta(1)-integrin bright) and TAC (beta(1)-integrin dim) enriched fractions between normal and psoriatic keratinocytes. Some of these were expected, such as mRNA levels of keratins 6 and 10 and of the Ki-67 antigen. Most remarkable were differences in phenotype of the psoriatic TAC compared with TAC from normal skin. These subpopulations also displayed striking differences following culture. Downregulation of markers associated with the regenerative phenotype (psoriasin, elafin, psoriasis-associated fatty acid binding protein) in cultured psoriatic dim cells in the absence of hyperproliferation suggests that proliferation and regenerative maturation are coupled. From these results, in combination with our earlier findings, we propose a model for epidermal growth control in which TAC play a crucial role.

The demand for large-scale gene expression analysis tools is on the rise now that several genomes have been sequenced. One of these tools, serial analysis of gene expression (SAGE), allows the qualitative as well as quantitative analysis of a large number of genes in a defined tissue or culture model. SAGE has already been successfully used to identify differentially expressed genes in normal physiological processes and pathological conditions. This chapter focuses on the SAGE protocol and its application to cultured human keratinocytes, and on MicroSAGE, an adapted protocol that allows the use of small amounts of mRNA from isolated epidermis or a skin biopsy.

Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide. We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ. Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments. Image analysis was used to determine growth characteristics of the colonies. We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation. The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation. Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different. Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis in transformed and tumor cell lines, but usually not in other cells. Here we present a study on the effect of TRAIL on cultured keratinocytes. It is shown that differentiated and undifferentiated keratinocytes undergo apoptosis after addition of TRAIL to the medium as determined by morphologic and biochemical criteria, such as cellular shrinkage and activation of caspases. The sensitivity for TRAIL differs greatly between undifferentiated and differentiating keratinocytes, however, with undifferentiated cells being much more susceptible to apoptosis. Commitment to terminal differentiation in the absence of TRAIL does not in itself induce apoptosis. In contrast to the promyelocytic cell line HL60, internucleosomal DNA fragmentation is not observed in keratinocytes, as assessed by flow cytometric analysis and agarose gel electrophoresis. Interestingly, the prime effector of DNA fragmentation, DNA fragmentation factor of 40 kDa (DFF40), is expressed in keratinocytes, yet internucleosomal cleavage fails to occur. Our data indicate that programmed cell death during keratinocyte differentiation is distinct from receptor-mediated apoptosis in response to a death ligand.

Elafin (also known as skin-derived anti-leukoproteinase/trappin-2) is an epithelial host-defense protein that is absent in normal skin but highly induced in keratinocytes of inflamed skin (e.g., psoriasis), in epidermal skin tumors, and after wounding. Previously, it was shown that in cultured keratinocytes, elafin expression is induced by serum or tumor necrosis factor-alpha, and that expression is suppressed by retinoids, dithranol, and p38 mitogen-activated protein kinase inhibitors. Here we have studied the regulation of elafin gene expression in epidermal keratinocytes at the molecular level. First we determined the transcription start site of the elafin gene and found that the elafin mRNA possesses an unusually short 5'-untranslated region. Using transient transfection of luciferase reporter constructs of the elafin promoter, we mapped a 440 bp region upstream of the translation start site that conferred high-level expression in keratinocytes, but not in A431 cells or cells of mesenchymal origin. We observed that the promoter constructs were not subjected to the same regulation as the endogenous elafin gene as these constructs were highly active independent of keratinocyte stimulation. When elafin promoter constructs were stably transfected in the HaCaT keratinocyte cell line, tumor necrosis factor-alpha inducible expression of both the endogenous elafin gene and the transgene was observed, suggesting that regulation of the elafin gene is also dependent on chromatin structure. We found, however, that a stably transfected 4 kb elafin promoter fragment did not confer retinoid sensitivity indicating that additional sequences are required for proper regulation. This study reveals the complex regulation of a gene that can be used as a paradigm for the specific differentiation program of activated epidermal keratinocytes.

Pharmacological treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of two or more of these actions. Potentially new drugs for treatment of psoriasis, which act on proliferation, can be identified by screening large compound libraries in a cell proliferation model that allows for characterization of drug effects on in vitro growth of normal human keratinocytes. High-throughput programs based on biological testing of diverse collections of compounds can rapidly identify leads for potential drug candidates in the treatment of psoriasis. In this study, we describe nonradioactive measurement of keratinocyte proliferation in the exponential growth phase in a 96-well format, using a sensitive deoxyribonucleic acid-binding dye to analyze drugs that are pharmacologically active in growth inhibition. Release of lactate dehydrogenase was used to exclude cytotoxic effects. We examined a number of compounds in a test range of 10(-7) to 10(-5) M, including known antipsoriatic drugs, and experimental drugs that are potentially useful in the treatment of psoriasis. We found strong concentration-dependent growth inhibition by dithranol, an antipsoriatic compound that is presumed to target the epidermal compartment. Methotrexate, cyclosporin A, and all-trans retinoic acid did not significantly affect proliferation at therapeutically relevant concentrations. The p38 mitogen-activated protein kinase inhibitor, SB220025, and curcumin, a natural phytochemical, inhibited keratinocyte proliferation at 10(-5) M. We conclude that this assay, in combination with the previously developed assays for psoriatic differentiation, provides a useful tool for identification of antipsoriatic drugs.

Oral reconstructions for cleft palate repair are often complicated by a shortage of mucosal tissue. This shortage causes scar tissue formation leading to impaired growth of the dento-maxillary complex. The overall aim of our research is to develop a substitute. which limits the iatrogenic effects of cleft palate surgery. This study describes the culture and characterization of mucosal substitutes containing keratinocytes. Epidermal and oral keratinocytes from a beagle dog were cultured on several skin-derived and collagen-based substrates. Oral keratinocytes cultured on the skin-derived substrates closely resembled normal oral epithelium of the dog. A multi-layered epithelium was formed showing parakeratosis, expression of cytokeratin 16 and the formation of a basement membrane. Epidermal keratinocytes cultured on the skin-derived substrates formed an epithelium which was similar to dog epidermis. In contrast, keratinocytes cultured on the collagen-based substrates invaded the substrate without the formation of a multi-layered epithelium. In conclusion, this study shows that oral canine keratinocytes cultured on skin-derived substrates exhibit a tissue organization that resembles normal oral mucosa. This type of mucosal substitute will therefore be used in further studies for implantation on the palate of beagle dogs. These studies might eventually lead to an improvement of cleft palate surgery in humans.

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Established treatments for psoriasis act ei-ther on hyperproliferation, inflammation, aberrant epidermal differentiation or a combination of these aspects of the disease. Potential new drugs for treatment of psoriasis or other disorders with abnormalities in epidermal differentiation can be identified by high-throughput screening of large compound libraries using surrogate markers for the disease. Here we describe a screening model to detect pharmacologically active drugs in two keratinocyte-based, 96-well culture models that use expression of cytokeratin 10 (CK10) and skin-derived antileucoprotease (SKALP)/elafin as markers for normal and psoriatic differentiation, respectively, and allow multiple parameters to be determined from a single well. In this model we tested a number of compounds in a pharmacological range from 10(-7) to 10(-5) M, including known antipsoriatic drugs, and experimental drugs that are potentially useful in the treatment of psoriasis. All-trans-retinoic acid, dithranol and the p38 mitogen-activated protein (MAP) kinase inhibitor SB220025 displayed a strong inhibitory effect on SKALP expression while cyclosporin A, dexamethasone, the vitamin D(3) derivative calcipotriol and the p38 MAP kinase inhibitor SB203580 showed only moderate inhibition. Methotrexate and dimethylfumarate did not affect the expression of SKALP. With respect to CK10 expression, all-trans-retinoic acid, calcipotriol, SB203580 and SB220025 exhibited strong inhibition while dithranol showed only moderate suppression of this normal differentiation marker. Expression levels of CK10 were not significantly affected by dexamethasone, methotrexate, cyclosporin A or dimethylfumarate. This model system parallels most, but not all, findings on the in vitro effect of known antipsoriatic drugs on keratinocytes. In addition, the model identifies p38 MAP kinase inhibitors as potent suppressors of differentiation-associated gene expression. Although further delineation and validation of this model is required, we conclude that the system is amenable to down-scaling and application as a high-throughput screen for differentiation-modifying compounds., (Copyright 2002 S. Karger AG, Basel)

Serial analysis of gene expression (SAGE) has been used for quantitative analysis of gene expression. We applied cluster analysis on multiple SAGE libraries derived from premalignant epidermal tissue (actinic keratosis), normal human epidermis, and cultured keratinocytes. The samples were obtained from skin biopsies without contamination by dermal tissue or blood. A total of 60,000 transcripts (tags) were analyzed. Two-way cluster analysis was applied to both the transcripts and the tissues, resulting in separation of the cultured cells from the epidermal samples, and clustering of many, presumably coregulated, genes. Two clusters of genes, strongly up-regulated in the tumor tissue compared with normal epidermis, were investigated in more detail. The differential expression of genes could be confirmed in actinic keratosis from four patients. Several of these genes have been previously associated with carcinogenesis or are likely to be important on the basis of their presumed function. Automated literature search tools show that a subgroup of these genes is coexpressed in other tissues and is part of an epidermal differentiation gene cluster on chromosome 1q21. We conclude that cluster analysis on large data sets uncovers clear partitions and correlations that could be confirmed by independent methods. We predict that these partitions will lead to biological interpretations that can be relevant for understanding the processes of carcinogenesis and tumor progression.

Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified.

Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4-8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFkappaB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations.

Keratinocyte gene expression was surveyed more comprehensively than before, by means of serial analysis of gene expression. A total of 25,694 tags derived from expressed mRNA, were analyzed in a model for normal differentiation and in a model where cultured keratinocytes were stimulated for a prolonged period of time with tumor necrosis factor-alpha, thus mimicking aberrant differentiation in the context of cutaneous inflammation. Serial analysis of gene expression revealed many transcripts derived from unknown genes and a large number of genes that are not known to be expressed in keratinocytes; furthermore, these data provide quantitative information about the relative abundance of transcripts, allowing the identification of differentially expressed genes. A major part of the identified transcripts accounted for genes involved in energy metabolism and protein synthesis. A large proportion of all transcripts (6%) corresponded to genes associated with terminal differentiation and barrier formation. Another highly expressed functional group of genes (2% of all transcripts) corresponded to proteins involved in host protection such as antimicrobial proteins and proteinase inhibitors. Three of these genes were not known to be expressed in keratinocytes, and some were upregulated after prolonged tumor necrosis factor-alpha exposure. Our data on expressed genes in keratinocytes are consistent with the known function of human epidermis, and provide a first step to generate a transcriptome of human keratinocytes.

Keratinocytes of inflamed epidermis (psoriasis, wound healing) are hyperproliferative and display an abnormal differentiation programme. This regenerative differentiation pathway is characterized by the induction of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6, CK16, CK17, and the proteinase inhibitor SKALP/elafin. In the study reported here we investigated the induction and regulation of SKALP expression as a marker for regenerative differentiation in epidermal keratinocytes. Various cytokines and growth factors known to be present in psoriatic epidermis were examined for their ability to induce SKALP gene expression in cultured human keratinocytes. Tumour necrosis factor-alpha (TNF-alpha) and serum were found to be potent inducers of SKALP expression at both the mRNA and the protein levels. SB202190 or SB203580, two specific p38 MAP kinase inhibitors almost completely blocked the induction of SKALP expression by TNF-alpha and serum. These results suggest that in keratinocytes, p38 activity is crucial for the induction of SKALP gene expression. These findings could be relevant for the elucidation of the mechanisms involved in normal and disturbed epidermal differentiation.

Secretory leukocyte protease inhibitor (SLPI) is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte-derived proteinases, has anti-HIV-1, antibacterial, and anti-fungal properties, and interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages. We now report that at both the mRNA and the protein level, SLPI shows inducible expression in a nonglandular epithelium. A weak expression of SLPI was found in the stratum granulosum of adult normal human epidermis; however, in lesional psoriatic epidermis and in migrating keratinocytes of healing wounds, a strong cytoplasmic staining was seen in the suprabasal keratinocytes. Remarkably, in the dermis adjacent to SLPI-expressing keratinocytes, SLPI was found extracellularly associated with elastin fibers, whereas the dermis in normal skin was negative. In cell culture, SLPI was hardly expressed in monolayers of proliferating keratinocytes. Differentiating cultures with a phenotype of normal skin expressed low levels of SLPI, whereas cultures with a regenerative/psoriatic phenotype expressed high levels. Functional studies with recombinant SLPI indicated that its antibacterial spectrum and potency are distinct from other anti-microbial peptides such as lysozyme and defensins. In view of the multiple functions of SLPI and the inducibility, we propose that it acts as an important first line defence mechanism in cutaneous injury.

Recently we showed that human epidermal keratinocytes express the extracellular matrix protein tenascin-C (TN-C) during wound healing, but not in normal adult skin. To gain further insight into the regulation of epidermal TN-C expression, we tested the effect of various stimuli on TN-C expression by cultured keratinocytes. Our results indicate that IL-4 is a very strong inducer of TN-C protein and mRNA expression in normal keratinocytes. Furthermore, TNFalpha and IFNgamma moderately increased TN-C expression. No other cytokines and growth factors that we tested, including various factors that stimulate TN-C expression in mesenchymal cells, significantly affected TN-C secretion by cultured keratinocytes. The regulation of TN-C expression in keratinocytes is distinct from that of fibronectin, since IL-4 and IFNgamma did not affect fibronectin expression in our experiments, and TNFalpha only slightly increased fibronectin levels. To investigate the role of cellular stress response pathways that can be activated by TNFalpha in the regulation of TN-C expression, we tested the effect of different inhibitors and an activator of these intracellular signalling cascades. The results show that the p38 MAP-kinase pathway is not involved in TNFalpha-induced TN-C expression in cultured keratinocytes. Activation of the JNK/SAPK-1 pathway by the addition of sphingomyelinase resulted in a dose-dependent increase of TN-C expression. TN-C expression by squamous carcinoma cell lines was differentially affected by the cytokines that stimulated TN-C expression in normal keratinocytes: TNFalpha again increased TN-C secretion, but IL-4 and IFNgamma had little effect. We conclude that there are distinct regulation mechanisms for TN-C expression in normal keratinocytes, tumor-derived keratinocytes and mesenchymal cells. The observation that TN-C is abundant in inflamed skin is a strong indication that inflammatory cytokines such as IL-4, TNFalpha and IFNgamma could also be involved in the regulation of epidermal TN-C expression in vivo.

We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on epidermal keratinocytes is qualitatively different from the other detergents tested, which might have implications for in vitro toxicology studies that use cell biologic parameters as a read-out. We would hypothesize that detergents cause skin injury by several mechanisms that include direct cellular toxicity, disruption of barrier function, and detergent specific effects on cellular differentiation, as demonstrated here for sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, and sodium laurate.

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes.

Tenascin-C is a large hexameric extracellular matrix glycoprotein that is expressed in a temporally and spatially restricted pattern associated with stromal-epithelial interactions. In adult human skin, the expression level of tenascin-C is low, but tenascin-C is abundantly present in the dermal compartment during embryogenesis and wound healing and in skin tumors. Herein we have investigated the cellular source of tenascin-C production in human skin, both in vivo and in vitro, by using immunohistochemistry, mRNA in situ hybridization, western blotting, and an enzyme-linked immunosorbent assay. In addition we studied the cell-matrix interaction between epidermal keratinocytes and purified tenascin-C. By using in vitro culture models, we found that keratinocytes not only synthesize and secrete tenascin-C but can also deposit tenascin-C in de-epidermized dermis in a pattern that is very similar to that in vivo. In vivo, during wound healing of normal human skin, we found tenascin-C extracellularly in the wound bed and also in a granular pattern within the neo-epidermis. By mRNA in situ hybridization, we could identify the basal migrated keratinocytes as the main source of tenascin-C in the early phase of wound healing. In the granulation phase, tenascin-C expression by the keratinocytes is downregulated. Cultured keratinocytes were found to adhere poorly to tenascin-C, and those that did adhere retained a rounded morphology. We conclude that human keratinocytes are a major source of tenascin-C during the early phase of wound healing, and we hypothesize that tenascin-C is unlikely to be an adhesive substrate for migrating keratinocytes.

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

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor beta 1 (TGF-beta 1) and (iii) restimulation from the G0-phase of the cell cycle. Bivariate analysis of lodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (Ts), total cell cycle duration (Tc), responsiveness to TGF-beta 1 and the kinetics for recruitment from G0. In psoriatic keratinocytes we found a lower amount of cell in S-phase and a shorter duration of G1, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators.

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

The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control.

In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1. It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.

Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8-10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study we performed a cell kinetic characterization of the growth and differentiation of human keratinocytes. Using a combination of immunohistochemical and flow cytometric techniques it was possible to obtain a detailed description of these processes. The proliferative activity of the cell cultures was analysed with flow cytometric techniques, measuring relative DNA content, iododeoxyuridine incorporation and the expression of the antigen recognized by Ki-67. In addition to a standard monolayer culture technique, cells were maintained in suspension. Under these conditions these cells were not capable of dividing, started to lose their nuclei, and the expression of differentiation-related proteins such as involucrin and filaggrin was induced, suggesting that the cells changed towards a differentiated phenotype. Binding of the antibody Ks8.12, recognizing keratins 13 and 16, occurred under all culture conditions, independent of cell density, and also in suspension, suggesting that it is a marker for abnormal differentiation rather than for hyperproliferation.