Identification of novel genes for secreted and membrane-anchored proteins in human keratinocytes.

Abstract

Both intercellular and intracellular signals are transduced primarily by interactions of secreted and/or membrane-anchored polypeptides, and they play a pivotal role in regulating proliferation, differentiation and apoptosis of keratinocytes within the epidermis. Despite recent identification of these polypeptides, it is likely that several important molecules remain undisclosed. To identify novel genes encoding secreted or membrane-anchored polypeptides expressed by human keratinocytes. We employed a signal sequence (SS) trap of a 5'-end-enriched cDNA library prepared from primary cultured human keratinocytes. Gene expression analysis was performed using Northern blotting. R Screening of 4018 cDNA clones yielded 82 positive clones (57 independent genes), most of which encoded SSs in their N-termini. Most of the positive clones were known genes registered in the GenBank database. Seven genes were identified in the EST database, four of which encoded novel membrane-anchored polypeptides with features of type I transmembrane proteins; the other three genes encoded novel non-type I transmembrane polypeptides. These EST genes were expressed differentially by keratinocytes subjected to low vs. high calcium concentrations and by basal vs. squamous cell carcinomas. Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.