Our previous work has established a role for the novel NADPH oxidase Nox4 in the regulation of vascular smooth muscle cell (VSMC) phenotype. We showed that Nox4 and its regulator, polymerase delta interacting protein II (Poldip2), are required for maintenance of differentiation marker gene expression, as well as the formation of smooth muscle a-actin based stress fibers and focal adhesion turnover. In this project, we wish to extend our understanding of the mechanisms by which Nox4/Poldip2 regulates both gene expression and the cytoskeleton, as these are universal effects that could explain the many roles of Nox4 in different cell types. We propose to pursue three specific aims. First, we will determine the molecular mechanisms underlying Nox4/Poldip2-mediated regulation of the paxillin homologue Hic-5 and microtubules and their role in focal adhesion turnover and differentiation marker gene expression. We hypothesize that Nox4 regulates Hic-5 either transcriptionally or via oxidation and targeting to the proteasome, and Nox4-mediated activation of leads to activation of mammalian diaphanous-2 (mDia2), a Rho effector that stabilizes microtubules. Second, we plan to define the role of Nox4-mediated p-actin oxidation in TGF-p-induced smooth muscle a-actin gene expression. We propose that Nox4/Poldip2 oxidizes p-actin, thus stabilizing F-actin filaments and promoting the translocation of the smooth muscle specific transcription factor, MRTF-A, to the nucleus, where it promotes gene expression. Finally, we will determine the role of Nox4/Poldip2 in cell cycle regulation in vitro and in vivo, as well as in regulating the balance between proliferation and redifferentiation of smooth muscle cells after injury. In this aim, we will use cultured cells to probe the mechanisms responsible for Nox4/Poldip2-induced G2/M arrest, focusing on the cell cycle phosphatases cdc25b and cdc2. In addition, we will use a femoral artery wire injury model in Poldip2 heterozygous mice to create a situation in vivo in which VSMCs are stimulated to proliferate in the presence of impaired Nox4 activity. Each of these aims is based on strong preliminary data and will help us to reach our overall goal of defining the role of Nox4 in VSMC function. Since very few molecular targets of Nox4 are known, these studies will greatly advance our understanding of Nox4 biology. If we are able to distinguish between Nox4 signaling to gene expression and Nox4-mediated regulation of the cytoskeleton, "smart" pharmacological interventions could be created to selectively interfere with particular functions of Nox4, while leaving other critical physiological pathways unaffected.