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Intracellular signaling pathways regulating integrin-mediated keratinocyte spreading and lamellipodia formation Alavian, Keyhan

Abstract

The process of wound healing involves formation of long cytoplasmic extensions by keratinocytes followed by the their migration into the wound. This process is regulated through intracellular pathways triggered by integrin-extracellular matrix interactions. Still, little is known about the intracellular pathways involved in formation of these "extended lamellipodia" in keratinocytes and their effects on cell migration. In this study, we used a number of kinase and phosphatase modulators to target various signaling pathways inside keratinocytes spreading on the wound provisional matrix, fibronectin. Our results showed that staurosporine (STP), a broad kinase inhibitor, leads to formation lamellipodia in keratinocytes at 10 nM and extended lamellipodia (E-Lam) at 50 nM. Cell spreading however was not concentration dependent since both STP concentrations increased cell spreading and migration by same amount (200%). Neither cell spreading or E-Lam formation involves modulation of phosphatidylinositol-3 kinase (PI-3K) or extended tyrosine phosphorylation since inhibitors against these proteins had no effect on STP-induced E-Lam formation and cell spreading. Actin and microtubule polymerization, however, are required for the formation of E-Lams. Western blotting experiments showed that keratinocytes treated with 50 nM STP exhibit twice the level of mitogen-activated protein kinase (MAPK) activation as keratinocytes treated with 10 nM STP. Immunostaining experiments with anti-tubulin antibodies demonstrated that microtubules (MTs) are localized at the tip of the extended lamellipodia when treated with 50 nM STP, suggesting that MTs may play an active role in E-Lam formation. Further experiments using inhibitory ocv and pi antibodies confirmed previous results that cell spreading and E-Lam formation is mediated through αvβl, α5βl, and αvβ6 integrins. Based on these findings, we speculate that differential activation of MAPK may play an active role in regulation of E-Lam formation involved in cell spreading and cell migration, by modulation of integrins binding-affinity, and organization of the cytoskeletal actin and microtubules.

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