alexa An in vitro analysis of mechanical wounding-induced ligand-independent KGFR activation.
Genetics & Molecular Biology

Genetics & Molecular Biology

Journal of Tissue Science & Engineering

Author(s): Li M, Firth JD, Putnins EE

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Abstract BACKGROUND: KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood. OBJECTIVES: We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism. METHODS: Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors. RESULTS: Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7\%), KGFR inhibitor SU5402 (70\%) and MAPK inhibitor PD98059 (57\%). CONCLUSION: Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration. This article was published in J Dermatol Sci and referenced in Journal of Tissue Science & Engineering

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