Comparison of gene expression profiles in human keratinocyte mono‐layer cultures, reconstituted epidermis and normal human skin; transcriptional effects of retinoid treatments in reconstituted human epidermis

Abstract

In order to validate a model for predictive screening of dermatological drugs, we used a customized cDNA macro-array system containing 475 skin-related genes to analyze the gene expression patterns in human keratinocytes from different origins: (1) normal human epidermal keratinocyte mono-layer cultures, (2) the commercially available SkinEthic reconstituted human epidermis model, and (3) biopsies of normal human epidermis. Few markers of those that were detected significantly in keratinocyte mono-layers or in reconstituted epidermis were undetected or detected at very low level in the normal epidermis biopsies. A comparative expression of more than 100 markers could be evidenced in both normal epidermis and reconstituted epidermis samples; however, only 90% of these were detected in keratinocyte mono-layers: expression of several terminal differentiation markers, such as filaggrin, loricrin, and corneodesmosin were strongly detected in normal epidermis and reconstituted epidermis, but were not significantly expressed in keratinocyte mono-layers. Under the experimental conditions described herein, the reconstituted human epidermis model was found to significantly reproduce the gene expression profile of normal human epidermis. Using the same methodology, we then investigated the effects of all-trans retinoic acid, 9-cis retinoic acid, all-trans retinol and a commercialized tretinoin-containing cream (Retacnyl) on the gene expression profiles of reconstituted human epidermis. According to the nature and the length of the treatments, more than 40 genes were found significantly modified. Among the genes whose expression was decreased, we found cytokeratins 1, 10, 2E, and 6B, several cornified envelope precursors, integrins alpha 3, alpha 6, beta 1, beta 4, some components of desmosomes, of hemi-desmosomes and of the epidermal basement membrane. Transcriptional upregulation was observed for keratins 18 and 19, autocrine and paracrine growth factors such as HB-EGF, IGF 1, PDGF-A, calgranulins A and B, interleukin-1 alpha and the other IL-1-related markers, type II IL-1 receptor and type I IL-1-receptor antagonist. Our results confirm most of the known effects of retinoids on human epidermis, but also give new insights into their complex pharmacological activity on skin. The reconstituted human epidermis used proves to be a highly predictive model for efficacy evaluation of skin-targeted compounds, such as retinoids.

IL-22 Inhibits Epidermal Differentiation and Induces Proinflammatory Gene Expression and Migration of Human Keratinocytes

“…Membranes were exposed for 4 days to a Molecular Dynamics Storm storage screen and scanned using a phosphorimager scanner (Molecular Dynamics Storm analyser; Amersham Biosciences). After local background substraction, an average signal intensity from duplicate spots was normalized for differences in probe labeling using the values obtained for housekeeping genes (24). For each gene, the IL-22-induced modulation was expressed as the relative expression value for stimulated vs control sample.…”

“…Modification of the keratinocyte gene expression profile induced by IL-22 was analyzed using a cDNA array specially designed for the study of skin cells and keratinocytes and consisting of 475 different cDNAs, spotted in duplicate, that correspond to proteins involved in cell structure, metabolism, extracellular matrix, adhesion, differentiation, signaling, signal transduction, apoptosis, and stress (24). Analysis of Atlas cDNA array membranes revealed an IL-22-dependent increase in the expression of the calcium-binding protein S100A8 (GenBank accession no.…”

“…DNase treatment, poly(A) ϩ RNA enrichment, 33 P-labeled cDNA probe synthesis, and purification and hybridization to custom Atlas array membranes (24) were performed according to Clontech's recommendations (Clontech). Membranes were exposed for 4 days to a Molecular Dynamics Storm storage screen and scanned using a phosphorimager scanner (Molecular Dynamics Storm analyser; Amersham Biosciences).…”

Soluble Form of Heparin-binding EGF-like Growth Factor Contributes to Retinoic Acid-induced Epidermal Hyperplasia

“…However, the effects of retinoids are often conflicting: retinoids can either stimulate or inhibit cell proliferation depending on the cell type affected or on the conditions used in cell cultivation (Amos and Lotan, 1990;Fisher and Voorhees, 1996;Rogers, 1997;Varani et al, 1989Varani et al, , 1991. Although the mechanism by which retinoids regulate the proliferation of epidermal cells is not completely understood, it has been shown that treatment of normal adult human skin and keratinocytes with tRA not only leads to thickening of the skin but also induces significant expression of several genes including HB-EGF (Bernard et al, 2002;Stoll and Elder, 1998;Varani et al, 2001). Induction of HB-EGF has also been reported in the skin of mice treated topically with tRA (Chapellier et al, 2002;Xiao et al, 1999).…”

Análise mecânica e histológica do tegumento facial com seqüela de queimadura após tratamento tópico com tretinoína

“…Destes, podem ser citados fatores de crescimento epidérmico como o fator de crescimento derivado de plaquetas (PDGF -platelet derived growth factor) e o fator transformador de crescimento beta (TGF-β), que, por sua vez, influenciam a síntese de colágeno pelos fibroblastos 48 .…”