ABSTRACT: In epidermis, granular keratinocytes are responsible for the synthesis of a great number of proteins of high tissue-specificity, and required for the epidermal barrier establishment and the regulation of the desquamation process. This project aims at characterizing the physiopathological function of genes implicated in these processes, and in the short term, defining their expression level in the course of keratinocyte differentiation. Pangenomic oligonucleotide microarrays are used to compare their expression between two cell populations enriched in basal or granular keratinocytes (T1 and T4 fractions resp.), obtained by successive incubations in trypsin solution of six independent normal human epidermis samples. Real-time quantitative PCR experiments were further performed to ensure the validity of the profiling for each candidate gene identified from microarrays data, and results of this multitechnique approach will be the object of a forthcoming publication. These results will be the basis for a better understanding of dermatological hyperkeratotic pathologies. Within epidermis, keratinocytes achieve a differentiation program culminating in cornification, a programmed cell death required for the establishment of the barrier function. A complex genetic regulatory network underlies the numerous biochemical modifications taking place in the last living cells, the granular keratinocytes (GKs), but very few transcription factors specific from this process were identified. Here we describe a large-scale, multitechnique approach performed on cells purified from normal human epidermis, and mainly focused on identification of these regulators. We performed microarray experiments to compare gene expression in cell fractions enriched in granular or basal keratinocytes. These data, with in silico promoter analysis for 52 differentiation markers, and exploitation of an EST library from GKs, were filtered according to gene products’ known or putative function and ESTs number in UniGene database, and allowed identification of 298 candidates out of 3,662 genes potentially specific from the last differentiation steps. Half were detected for the first time in epidermis and quantified by quantitative real-time PCR. These genes are linked to various aspects of GKs function, including structural components, lipid metabolism and secretory apparatus, cell signaling, and regulation of transcription. Forty-nine new differentiation-associated genes were identified, and 38 transcription factors in 92 were positively or negatively regulated in the course of differentiation, therefore possibly involved in the final differentiation of GKs. These data give new leads to have a global view of the network involved in epidermal barrier function, and allow in the future a better understanding of several pathologies, including genodermatoses of unknown aetiology. Keywords: Cell type comparison Six independent samples of normal human epidermis were used for this study. For three samples, technical replicates (at the labelling step) were performed (ie AQQ003, AQQ005 and AQQ011 slides are dye-swaps of AQQ002, AQQ004 and AQQ010, respectively). Data from each pair of “swap arrays” were merged and considered as coming from three simple arrays (named AQQ002dw, AQQ004dw and AQQ010dw) for statistical analysis. For each slide, T4 is the test sample (total RNA from granular keratinocytes), and T1 the reference sample (total RNA from basal keratinocytes of the same subject), and both come from the same biological sample.
