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Migratory and Proliferative Effects of Kgf Are Mediated by Erk 1/2 Mapkinase Pathway and Ccaat/Enhancer Binding Proteins

Piyush Koria and Stelios T. Andreadis. SUNY at Buffalo, 916 Furnas Hall, Amherst, NY 14260

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. It is produced by stromal cells but effects epithelial cells. It plays an important role in epithelial morphogenesis and its expression is highly up-regulated during wound healing. KGF promotes growth and migration of human keratinocytes on fibronectin. Keratinocytes bind to fibronectin through integrin alpha-5 beta-1, which is not expressed in normal skin but it is highly up-regulated by keratinocytes of the migrating epidermis during wound healing. Since KGF and integrin alpha -5 have similar patterns of expression we hypothesized that KGF may induce expression of integrin alpha-5 and investigated the signaling pathway and transcription factors that mediate this response. Using a scratch wound model system, we showed that KGF increased wound healing in a dose dependent manner and that blocking integrin alpha-5 decreased the KGF-mediated enhancement of migration. We further found that KGF increased the mRNA and protein of this integrin in primary keratinocytes, A431 cells and most important in fully stratified bioengineered skin. Using MEK inhibitors PD98059 and U0126 we show that KGF-mediated alpha-5 upregulation was dependent on ERK 1/2 MAPK pathway. Promoter studies indicated that KGF increased alpha-5 transcription through the c/Ebp transcription factor biding site of the alpha-5 promoter. Furthermore, KGF induced nuclear localization of c/Ebp beta and delta isoforms in engineered skin equivalents, suggesting their role in alpha-5 transcription. KGF also decreased the expression of c/Ebp alpha in the basal and suprabasal layers, suggesting that KGF-mediated over-proliferation in these compartments might be mediated through c/Ebp alpha. Indeed, overexpression of c/ebp alpha in A431 cells completely blocked KGF-induced hyper-proliferation in A431 cells. Our results indicate that KGF-induced migration and proliferation may be mediated via two distinct isoforms of the c/Ebp transcription factor family. We are currently working in identifying which isoform (c/Ebp beta or delta) is important for alpha-5 gene transcription using siRNA and dominant negative approaches. An understanding of the molecular interactions of KGF with integrins may help us to design therapeutics targeted towards improved wound healing and engineer better skin substitutes for treatment of burns and chronic wounds.