Vascular smooth muscle cell (SMC) differentiation is a very important process during vasculogenesis and angiogenesis, and it is well recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis. However, we are just beginning to understand the transcription mechanisms that regulate SMC differentiation, and we know very little about the signaling mechanisms that regulate this process. We previously showed that the small GTPase, RhoA, was an important regulator of SMC differentiation marker gene expression, and we have extended and confirmed these studies by demonstrating that the strong RhoA agonist, sphingosine-1 phosphate, stimulates SMC-specific transcription, that the RhoA effectors mDia1 and mDia2 play a critical role in this process, and that the effects of RhoA/mDia signaling are mediated by nuclear localization of the myocardin-related transcription factors (MRTFs). The goals of the current proposal are to further define the RhoA-dependent signaling mechanisms that regulate SMC differentiation marker gene expression and to study the involvement of RhoA signaling during SMC differentiation in vivo.
Our specific aims are as follows;1) To determine the contribution of RhoA signaling to SMC differentiation of in vivo. We will express constitutively active and dominant negative forms of RhoA in SMC and presumptive SMC using tamoxifen-inducible SM MHC and Wnt1 Cre mice. We will examine mice for changes in SMC differentiation during development and in adults following vessel injury. 2) to identify the RGS- RhoGEFs that regulate RhoA activity and SMC differentiation marker gene expression in SMC. We will use a well-established retroviral-mediated siRNA approach to knock down LARG, p115, and PDZ RhoGEF in rat aortic SMC emphasizing effects on RhoA activation, SMC differentiation marker gene expression, SMC migration, and nuclear localization of the MRTFs. 3) To identify the molecular mechanisms that regulate mDia2 function in SMC. We will test our hypothesis that phosphorylation of mDia2 by Rho-kinase enhances mDia2 activity, and we will examine mDia2's role in the nucleus in regard to nuclear structure, CRM-1- dependent nuclear export, and the transcriptional activity of the MRTFs. Completion of these aims should lead to a better understanding of the regulation of SMC differentiation.

Public Health Relevance

Vascular smooth muscle cell differentiation is a very important process during the development of blood vessels and it is well recognized that alterations in this process play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis. Our proposal examines the molecular mechanisms that regulate smooth muscle differentiation and should help to identify therapeutic targets for the treatment of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL070953-09
Application #
8212008
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Olive, Michelle
Project Start
2002-06-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
9
Fiscal Year
2012
Total Cost
$326,413
Indirect Cost
$103,663
Name
University of North Carolina Chapel Hill
Department
Pathology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Staus, Dean P; Weise-Cross, Laura; Mangum, Kevin D et al. (2014) Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription. Am J Physiol Heart Circ Physiol 307:H379-90
Rozenberg, Julian M; Tesfu, Daniel B; Musunuri, Srilaxmi et al. (2014) DNA methylation of a GC repressor element in the smooth muscle myosin heavy chain promoter facilitates binding of the Notch-associated transcription factor, RBPJ/CSL1. Arterioscler Thromb Vasc Biol 34:2624-31
Staus, Dean P; Taylor, Joan M; Mack, Christopher P (2011) Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain. Biochem J 439:57-65
Staus, Dean P; Blaker, Alicia L; Medlin, Matt D et al. (2011) Formin homology domain-containing protein 1 regulates smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol 31:360-7
Doherty, Jason T; Lenhart, Kaitlin C; Cameron, Morgan V et al. (2011) Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating protein (Rho-GAP), GRAF1. J Biol Chem 286:25903-21
Mack, Christopher P (2011) Signaling mechanisms that regulate smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 31:1495-505
Cheng, Zhaokang; Sundberg-Smith, Liisa J; Mangiante, Lee E et al. (2011) Focal adhesion kinase regulates smooth muscle cell recruitment to the developing vasculature. Arterioscler Thromb Vasc Biol 31:2193-202
Medlin, Matt D; Staus, Dean P; Dubash, Adi D et al. (2010) Sphingosine 1-phosphate receptor 2 signals through leukemia-associated RhoGEF (LARG), to promote smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 30:1779-86
Doherty, Jason T; Conlon, Frank L; Mack, Christopher P et al. (2010) Focal adhesion kinase is essential for cardiac looping and multichamber heart formation. Genesis 48:492-504
Blaker, Alicia L; Taylor, Joan M; Mack, Christopher P (2009) PKA-dependent phosphorylation of serum response factor inhibits smooth muscle-specific gene expression. Arterioscler Thromb Vasc Biol 29:2153-60

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