Smooth muscle cell gene expression controls the development and fate of vascular and non-vascular smooth muscle cells. In adult vascular smooth muscle cells (VSMC), the pattern of gene expression determines whether the cells exist in a contractile phenotype that is responsible for the control of vascular tone, or in a synthetic phenotype that is responsible for the repair of vessels in response to injury. The mechanism of VSMC-specific gene expression is a subject of interest since an understanding of the molecular control of this process may lead to a better understanding of the genesis of vascular disorders. One VSMC-specific transcriptional mechanism that has been defined is the serum response factor (SRF)-myocardin dependent regulation of VSMC-specific gene promoters. SRF-myocardin dependent gene regulation is modulated by many factors including the Ternary Factor Complex protein, Elk-1. Elk-1 is regulated by cellular signaling pathways such as growth factor-dependent phosphorylation and the small ubiquitin-like modifier protein (SUMO). Post-translational modification of Elk-1 by phosphorylation and sumoylation have been shown to alter SRF nuclear import and SRF-myocardin dependent mRNA expression. Likewise, the RhoA-Rho kinase signaling is also important for VSMC-specific gene expression because the RhoA pathway is involved in nuclear import of SRF. The nitric oxide (NO)-cGMP pathway stimulates VSMC-specific gene expression. Our laboratory has shown that the downstream mediator of NO-cGMP signaling, cGMP-dependent protein kinase (PKG), stimulates VSMC-specific gene expression but the mechanisms are undefined. We have obtained preliminary evidence that indicates that PKG stimulates sumoylation of Elk-1 in VSMC, and several laboratories have suggested that NO-cGMP signaling may be involved in the regulation of the RhoA-Rho kinase pathway. Both effects of PKG, Elk-1 SUMOylation and RhoA signaling, may shed light on the mechanism by which NO signaling increases VSMC-specific gene expression. This proposal is a basic science investigation to define the possible mechanisms by which PKG regulates VSMC-specific gene expression. The hypothesis is that PKG stimulates Elk-1 sumoylation, an effect which de-represses the action of Elk-1 on SRF/myocardin-dependent gene expression, and enhances RhoA signaling in cultured VSMC. These effects, in turn, stimulate SRF nuclear import and SRF-myocardin stimulated gene expression leading the cells to assume a contractile phenotype.
The specific aims are to (1) determine the mechanism by which PKG increases SRF-dependent transcription via regulation of Elk-1 in rat aortic VSMC, and (2) to determine the mechanism by which PKG interacts with RhoA signaling to stimulate SRF-dependent gene expression. ? ?
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