Control of adhesion-dependent SMC plasticity during coronary and outflow tract de

描述(由申请人提供)：对平滑肌细胞(SMC)的生长、迁移和分化的精确调控对于血管的正常发育是必要的，而这些过程的控制缺陷导致了几种突出的先天性和成人型心血管疾病的进展。大量研究表明，这些SMC功能受生长因子和细胞外基质(ECM)-整合素相互作用的调节，而非受体酪氨酸激酶、粘着斑激酶(FAK)的激活在这些信号通路中起关键作用。我们以前已经证明，FAK的内源性抑制物FRANK选择性地在SMC中表达，尤其在管道血管中观察到高水平的表达，并且在出生后血管发育和血管损伤后，FRANK的表达显著上调。这些研究表明，SMC中的整合素基质信号是独一无二的，对FAK活性的精确调节在血管发育和血管损伤修复过程中至关重要。事实上，我们已经证明，在WNT-1或NKX2.5来源的SMC中，FAK的条件失活(通过同源重组)导致了与出生后生活不相容的持续性动脉干。由于主-肺动脉间隔涉及对几个SMC过程的动态控制，我们继续使用各种功能丧失/获得的方法来研究FAK和FRANK在SMC中的作用。基因缺失抑制FAK活性对细胞生长或ERK激活影响不大。然而，它强烈地抑制了PDGF-BB介导的细胞极化和迁移，这一效果可能是由于小GTP酶RAC-1的缺陷激活所致。有趣的是，我们的体外和体内模型的结果也表明FAK活性和SMC分化之间存在强烈的负相关(通过SMC分化标记基因的表达来评估)。为了进一步阐明这些反应中涉及的FAK依赖机制，我们在酵母双杂交筛选中鉴定了LIM结构域适配蛋白Leupasin，以寻找在SMC中表达的FAK相互作用蛋白。我们利用siRNA介导的方法从SMC中去除亮蛋白，这些研究表明亮蛋白对SMC的趋化是必不可少的。我们还发现了一个有趣的、潜在的重要发现，即亮蛋白在焦点粘连和细胞核之间穿梭，这一过程受到FAK信号的调控。我们还证明了亮蛋白的异位表达上调了多个SMC分化标志基因；下调了亮蛋白的表达抑制了SMC分化标志基因的表达；亮蛋白在体内与SMα-肌动蛋白启动子相互作用；以及亮蛋白与SRF和强大的SRF辅助因子myocardin发生了物理和功能上的相互作用。在这项研究中，我们试图确定FAK如何在血管形态发生过程中调节SMC的表型，并确定FAK和亮蛋白改变SMC运动和分化的确切机制(S)。我们推测亮蛋白整合了这些不同的SMC功能，并通过靶向运动细胞前沿的RAC-1激活来调节迁移，并通过调节SMC分化标记基因启动子上功能性SRF转录因子复合体的形成来调节分化。 与公共健康相关：精确微调的平滑肌细胞生长对于血管的正常形成和功能是必要的，也在损伤后的血管修复中发挥作用。我们试图研究细胞外基质在调节血管形成和愈合反应中的作用。这里提出的实验将使用细胞和遗传方法的组合。

DESCRIPTION (provided by applicant): 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.