Sphingosine-1-phosphate and the RGS RhoGEFs regulate vascular smooth muscle phenotype Public Deposited

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  • March 21, 2019
Creator
  • Medlin, Matthew Douglas
    • Affiliation: School of Medicine, Department of Pathology and Laboratory Medicine
Abstract
  • The regulation of smooth muscle cell (SMC) differentiation is critical during vascular development, and perturbations in this process contribute to a number of cardiovascular pathologies including atherosclerosis, hypertension, and restenosis. We have shown that activation of RhoA by sphingosine-1 phosphate (S1P) stimulates SMC-specific gene expression by promoting the nuclear localization of the myocardin-related transcription factors (MRTFs). The aim of this dissertation is to dissect the precise mechanisms by which S1P regulates SMC phenotype. Using a combination of receptor-specific agonists and antagonists we identified S1P2 as the driving S1P receptor sub-type that regulates SMC-specific promoter activity and differentiation marker gene expression in primary SMC cultures. In addition, over expression of Gα12 or Gα13 increased SMC specific transcription, a result in excellent agreement with the known G-protein coupling properties of S1P2. Given previous studies on the interaction of Gα12/13 with the RGS subfamily of RhoGEFs (LARG, PRG, P115), we hypothesized that one or more of these RhoA activators was important in S1P-mediated SMC differentiation. While expression of each of the RGS RhoGEFs activated SMC specific transcription, LARG exhibited the most robust effect invoking a 10 to 15 fold increase SM22 and SM α-actin promoter activity. LARG expression also resulted in increased stress fiber formation and MRTF-A nuclear localization. Importantly, siRNA-mediated depletion of LARG (by approximately 90%) inhibited activation of RhoA by S1P and also inhibited the effects of S1P on endogenous SMC differentiation marker gene expression and SMC specific promoter activity. Finally, knockdown of LARG promoted SMC migration as measured by scratch wound and transwell assays. These findings indicate that stimulation of RhoA activity by S1P2-dependent activation of LARG plays a critical role in the regulation SMC phenotype. Interestingly P115 RhoGEF appears to regulate SMC migration in opposition to LARG. Despite the importance of RGS RhoGEF signaling little is known about their regulation. This thesis will explore the mechanisms regulating RGS RhoGEFs mediated RhoA activity and how differential RhoA activation may help modulate SMC phenotype. In conclusion these studies have improved our understanding the very complex means by which S1P mediated signaling regulates SMC phenotype and by extension normal and pathological vascular development.
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology and Laboratory Medicine."
Advisor
  • Mack, Christopher P.
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  • Chapel Hill, NC
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  • Open access
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