Keratinocytes from intact, uninjured skin express specific patterns of genes that promote proliferation and differentiation. In injuries that disrupt the basement membrane, wound edge keratinocytes contact dermal extracellular matrix (ECM) and are activated to undergo global changes in gene expression, switching from a proliferation/differentiation program to one that supports re-epithelialization and repair of the tissue defect. The factors that induce keratinocyte activation following injury, whether soluble or matrix, remain to be fully elucidated. Type I collagen, an abundant dermal ECM protein, may play an important role in keratinocyte activation in wounds that disrupt the basement membrane. Indeed, when keratinocytes from intact skin are plated onto fibrillar type I collagen in vitro they are stimulated to express collagenase-1, a matrix metalloproteinase expressed invariantly by activated keratinocytes at the wound edge that is essential for migration across the denuded dermis. Differential display reverse transcription PCR was used to identify other activation-specific genes that are modulated in keratinocytes following contact with collagen. Three distinct classes of transcripts were identified: (1) known genes associated with keratinocyte differentiation, (2) known genes not previously described in skin, and (3) unknown, novel transcripts. These data indicate that keratinocytes are stimulated to express a number of transcripts following contact with type I collagen and suggest that this cell:ECM interaction serves as a critical determinant for cell activation upon wounding. cDNA clone GAP4O1, a novel transcript markedly induced (20-fold) following collagen contact, was sequenced and found to have 100% identity to nucleotides 10237-10379 of human chromosome 17 clone hCIT.22-K-21. Subsequent amplification and translation of the full-length cDNA revealed epsin 3, a novel 632 amino acid protein closely related to, but distinct from members of the epsin protein family. Epsins 1 and 2 are constitutively and ubiquitously expressed proteins that function as molecular bridges require for clathrin-mediated endocytosis. Interestingly and in contrast to epsins 1 and 2, we found that epsin 3 is expressed exclusively and invariantly by collagen-activated keratinocytes, suggesting a novel role for this transcript during epidermal repair. Moreover, we determined that similar intracellular signaling mechanisms were required for collagen induction of collagenase-1 and epsin 3, suggesting a common signaling pathway leading to induction of both genes following matrix contact. -The objectives of this proposal are: 1) to localize epsin 3 expression in vivo in wounded and pathologic tissues by in situ hybridization and immunohistochemistry, and 2) to further characterize the molecular mechanisms regulating matrix-induced epsin 3 expression in activated keratinocytes. We anticipate that the data generated from these proposed studies will significantly contribute to understanding the role of altered cell:matrix interactions in epidermal wound repair and provide sufficient preliminary data for an R01 research proposal.
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