Primary mouse keratinocytes provide a well-characterized model system for control of epithelial cell growth, differentiation and transformation. The role of Rho/Rac GTPases in this system has been largely unexplored. In the present proposal we will address this question with a specific focus on the role of Rho and its downstream effectors. Our main working hypothesis is that Rho signaling plays an important role in the coordination of the keratinocyte growth/differentiation process. In particular, the following specific aims will be pursued: I) We will test the hypothesis that Rho signaling plays an important role in the earliest structural changes associated with differentiation, specifically the establishment of cell/cell adhesion. This hypothesis will be tested with primary keratinocytes under growing versus differentiating conditions. Rho function will be perturbed by treatment with the Rho-inactivating C3 toxin as well as adenoviral-mediated expression of a dominant negative RhoN 19 and a constitutively active RhoVl4 mutant. The molecular mechanisms that underlie the involvement of Rho in cell/cell adhesion will be evaluated, including the role of specific Rho effectors and a cross-talk with other signaling pathways. II) We will test the hypothesis that Rho signaling exerts a broader role ii keratinocyte growth/differentiation control which may extend to cell cycle control and/or transcription of differentiation-related genes. This hypothesis will be tested by the analysis of keratinocytes in which Rho activity is suppressed or enhanced by adeno-RhoNl9 or adeno-RhoVl4 expression, respectively. The molecular mechanisms that underlie changes in cell cycle control and/or expression of differentiation-related genes will be evaluated, including the role of specific Rho effectors and a cross-talk with other signaling pathways. III) We will test the hypothesis that Rho signaling plays an important function in control of keratinocyte growth/differentiation and transformation in vivo. This hypothesis will be tested by infection of cultured keratinocytes with recombinant retroviruses expressing the dominant negative RhoNl9 as well as the constitutively active RhoVl4 mutants. Cells will be tested for their growth/differentiation and tumorigenic behavior after skin reconstitution experiments back onto mice, under conditions where well stratified and differentiated epidermis is formed, or keratinocyte tumors are induced.
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