(Verbatim from the application): The proliferative response of vascular smooth muscle cells (VSMCs) plays a key role in the recovery of blood vessels from injury as well as in atherosclerotic lesion formation and postangioplasty restenosis. The long-term goal of this research is to understand pathways important for transmission of proliferative signals from the cytoplasm to the nucleus of VSMCs. A relationship between Ca2+ and proliferation has been implicated, but the mechanism(s) have not been identified. In addition, whereas transmission of Ca2+ and mitogenic signals across the nuclear membrane is necessary to elicit specific changes in gene expression, the regulation of signaling in the cytoplasm versus the nucleus is not known. We propose a novel mechanism for Ca2+-mediated gene transcription in smooth muscle, in which Ran-mediated nuclear transport is central to the signaling pathway leading from a rise in [Ca2+]i to phosphorylation of the transcription factor CREB. The goal of this proposal is to define compartmentalization of Ca2+-mediated signaling molecules in VSMCs as they relate to phosphorylation of CREB and transcription through the Ca2+ and cAMP response element (CRE). Specifically, we will pursue the following Aims: 1) To elucidate the role of intracellular Ca2+ stores and nuclear Ca2+ in CREB activation pathways; 2) To explore the mechanisms and structural elements regulating nuclear import of CREB; 3) To understand the function that nucleocytoplasmic shuttling of CREB has in both its phosphorylation and capacity to mediate transcription through the CRE; Methods will include immunofluorescence to detect phosphorylated CREB, in vitro and live cell nuclear transport assays, Ca2+ imaging, protein interaction assays and CRE-luciferase reporter assays. The combination of in vitro molecular approaches and live cell transport models will drive support for new paradigms in the regulation of CREB-mediated gene transcription by Ca2+ and the Ran GTPase in VSMCs.
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