Precise regulation of smooth muscle cell (SMC) growth, migration, and differentiation is necessary for proper vascular development, and defective control of these processes contributes to the progression of several prominent congenital and adult onset cardiovascular diseases. Extensive studies indicate that these SMC functions are regulated by growth factors and extracellular matrix (ECM)-integrin interactions and that activation of the non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) plays a critical role in these signaling pathways. We have previously demonstrated that an endogenous inhibitor of FAK, termed FRNK, is selectively expressed in SMC with particularly high levels observed in conduit blood vessels and that FRNK expression is dramatically up-regulated during post-natal vascular development and following vessel injury. These studies suggested that integrin matrix signaling in SMC was unique and that precise regulation of FAK activity was critical during vascular development and vessel injury repair. Indeed we have demonstrated that conditional inactivation of FAK (by homologous recombination) in wnt-1 or nkx2.5-derived SMC led to persistent truncus arteriosus that was incompatible with post-natal life. Since aorticopulmonary septation involves dynamic control of several SMC processes, we have continued to study the role of FAK and FRNK in SMC using a variety of loss/gain of function approaches. Inhibition of FAK activity by genetic deletion had little effect on cell growth or ERK activation. However, it strongly inhibited PDGF-BB-mediated cell polarization and migration, an effect likely due to defective activation of the small GTPase Rac-1. Interestingly, results from our in vitro and in vivo models also indicated a strong inverse correlation between FAK activity and SMC differentiation (as assessed by SMC differentiation marker gene expression). In an attempt to further delineate the FAK-dependent mechanisms involved in these responses, we identified the LIM domain adapter protein, leupaxin, in a yeast two-hybrid screen for FAK interacting proteins expressed in SMC. We utilized siRNA-mediated approaches to deplete leupaxin from SMC and these studies revealed that leupaxin was essential for SMC chemotaxis. We also made the interesting and potentially important discovery that leupaxin shuttles between focal adhesions and the nucleus and that this process was regulated by FAK signaling. We also demonstrated that ectopic expression of leupaxin up-regulated multiple SMC differentiation marker genes;that knock-down of leupaxin attenuated SMC differentiation marker gene expression;that leupaxin interacted with the SM a-actin promoter in vivo;and that leupaxin interacted physically and functionally with SRF and the powerful SRF co-factor, myocardin. In this proposal we seek to determine how FAK regulates SMC phenotype during vascular morphogenesis and to identify the precise mechanism(s) by which FAK and leupaxin alter SMC motility and differentiation. We hypothesize that leupaxin serves to integrate these diverse SMC functions and regulates migration by targeting Rac-1 activation to the leading edge of motile cells and differentiation by regulating the formation of a functional SRF transcription factor complex on SMC differentiation marker gene promoters.

Public Health Relevance

Precisely fine-tuned growth of smooth muscle cells is necessary for proper formation and function of blood vessels and also plays a role in repairing blood vessels after injury. We seek to investigate the role of extracellular matrix in the regulation of blood vessel formation and healing responses. Experiments proposed herein will use a combination of cellular and genetic approaches.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular Differentiation and Development Study Section (CDD)
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Schramm, Charlene A
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Weise-Cross, Laura; Taylor, Joan M; Mack, Christopher P (2015) Inhibition of Diaphanous Formin Signaling In Vivo Impairs Cardiovascular Development and Alters Smooth Muscle Cell Phenotype. Arterioscler Thromb Vasc Biol 35:2374-83
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
Cheng, Zhaokang; DiMichele, Laura A; Rojas, Mauricio et al. (2014) Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.). J Mol Cell Cardiol 67:1-11
Lenhart, Kaitlin C; Becherer, Abby L; Li, Jianbin et al. (2014) GRAF1 promotes ferlin-dependent myoblast fusion. Dev Biol 393:298-311
Bai, Xue; Lenhart, Kaitlin C; Bird, Kim E et al. (2013) The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension. Nat Commun 4:2910
O'Neill 4th, Thomas J; Mack, Christopher P; Taylor, Joan M (2012) Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest. J Mol Cell Cardiol 53:156-64
Zajac, Britni; Hakim, Zeenat S; Cameron, Morgan V et al. (2012) Quantification of myocyte chemotaxis: a role for FAK in regulating directional motility. Methods Mol Biol 843:111-23
Medlin, Matt D; Taylor, Joan M; Mack, Christopher P (2012) Quantifying sphingosine-1-phosphate-dependent activation of the RhoGTPases. Methods Mol Biol 874:89-97
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

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