TARGETING EPITHELIAL CELL MIGRATION TO ACCELERATE WOUND HEALING

Successful wound healing requires the recruitment and migration of distinct cell types to the wound followed by re-epithelialization of the surface of injured tissue. Interventions that enhance the migration of effector cells to the site and temporally increase epithelialization can be clinically relevant. The recent identification of the specific proteins required for human epithelial cell migration by siRNA-mediated reduction of over 1000 specific proteins demonstrated that a limited number of protein pathways, with their constituent members (32 proteins in total), are required for successful epithelial cell migration and in vitro wound closure. The group of cell attachment and adhesion molecules that are necessary for this cell migration includes all four members of the RIPP complex, consisting of the proteins Rsu1, Integrin Linked Kinase (ILK), PINCH, and Parvin. Previous studies demonstrated a role for RIPP complex in fibrosis, but the regulation of complex formation and the mechanism by which the complex functions to control migration have not be elucidated. Relevant to this proposal, there is no information available concerning the role of the RIPP complex in wound healing. ILK binds to the cytoplasmic domain of the beta integrins via its carboxyl terminal domain; the other members of the complex are found in a complex with ILK. In addition, the correct association of these proteins in a functional complex depends on their phosphorylation by serine threonine kinases of the protein kinase C (PKC) family, suggesting that they may serve as 'druggable' targets for the control of epithelial cell closure of wounds. We will test the hypothesis that epithelial cell migration can be temporally regulated by the association of RIPP complex proteins into a functional association. In addition, we will determine if the process can be enhanced or inhibited by modulating the levels of the RIPP complex proteins as well by regulating their serine and threonine phosphorylation.Initially, we will determine the role of serine and threonine phosphorylation of RIPP complex proteins on human skin epithelial cell and primary keratinocyte migration in an in vitro assay. This aim will determine the contribution of kinase-induced phosphorylation events to the rate of cell migration and wound closure in a controlled in vitro assay. Cells will be exposed to chemical inducers and inhibitors of PKC, and RIPP protein phosphorylation will be followed by mass spectrometry in conjunction with assessment of the effect on cell migration. The effect of specific RIPP protein phosphorylation will be tested by monitoring migration of cells expressing the relevant phosphorylation site mutants following siRNA-mediated knockdown of corresponding endogenous proteins. The next aim will determine the efficacy of targeting this complex as a mechanism to regulate the rate of epithelial cell migration during wound closure. This will be accomplished by modulating the specific levels and the phosphorylation of RIPP complex proteins in an in vivo mouse model of wound healing. Elevated expression of specific RIPP complex proteins, including the relevant phosphorylation site mutants, will be used for in situ infection of dermal epithelial cells in a mouse punch wound assay. The reduction of specific RIPP proteins in wounds will be accomplished by introduction of siRNA in gels to punch wounds at specific times post wounding. The effects of RIPP expression on wound closure will be followed over time.