Dissertation/Thesis Abstract

Regulation of smooth muscle -specific transcription by serum response factor and formin homology domain containing protein 1
by Blaker, Alicia L., Ph.D., The University of North Carolina at Chapel Hill, 2008, 149; 3315719
Abstract (Summary)

The regulation of vascular smooth muscle cell (SMC) differentiation is important during vasculogenesis, angiogenesis, and cardiovascular diseases, such as atherosclerosis and restenosis. Previous studies have shown that SMC differentiation marker gene expression is regulated by serum response factor (SRF) and the myocardin family of SRF co-factors (myocardin and the myocardin-related factors, MRTF-A and MRTF-B). A major goal of the current studies was to identify post-translational modifications of SRF that regulate SMC-specific gene expression. By screening phosphorylation deficient and mimetic mutations in SRF -/- ES cells, I identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression. In vitro and in vivo labeling studies demonstrated that T159 was phosphorylated by protein kinase PKA, and results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to the CArG elements present within the promoters of the SMC-specific genes. Based upon the identification of Ubc9 in a yeast-two-hybrid screen for SRF binding proteins, I also tested the role of sumoylation on SRF activity. In vitro sumoylation assays identified K147 as the major SRF sumoylation site, but a sumoylation deficient K147R mutation had no effect on SRF-dependent SMC-specific gene expression.

Our lab has also demonstrated that MRTF nuclear localization and activity is regulated by changes in actin dynamics, and a second goal was to determine whether the diaphanous formin, FHOD1, played a significant role in this process. Using RNAi techniques I demonstrated that FHOD1 was important for SMC differentiation marker gene expression in 10T1/2 and that over expression of a constitutively active version of FHOD1 strongly up-regulated SMC-specific promoter activity. Additional studies showed that phosphorylation of FHOD1 in the diaphanous auto-regulatory domain may contribute to FHOD1 activation and that FHOD1-mediated actin polymerization in the nucleus may be important for FHOD1's effects on MRTF activation.

Taken together, my results indicate that PKA-mediated phosphorylation of SRF and FHOD1-mediated actin polymerization regulate SMC-transcription providing two novel signaling mechanisms for the control of SMC phenotype. Future experiments extending these findings should lead to a better understanding of SMC's role in cardiovascular disease and to targets for treating these conditions.

Indexing (document details)
Advisor: Mack, Christopher P.
Commitee: Caron, Kathleen M., Faber, James E., Majesky, Mark W., Sealock, Robert
School: The University of North Carolina at Chapel Hill
Department: Cell & Molecular Physiology
School Location: United States -- North Carolina
Source: DAI-B 69/07, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Cellular biology, Pathology, Anatomy & physiology
Keywords: Formin homology domain-containing protein, Serum response factor, Smooth muscle-specific transcription
Publication Number: 3315719
ISBN: 9780549671220
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