Smooth muscle adhesion and plasticity in coronary and outflow tract development

冠状动脉和流出道发育中的平滑肌粘附和可塑性

• 批准号: 8440760
• 负责人: Joan M Taylor
• 金额: $ 31.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440760
• 关键词: 3-Dimensional; Actins; Adhesions; Adhesives; Adult; Aorta; Attenuated; Binding; Biological Assay; Biological Models; Blood Vessels; Cardiovascular Diseases; Cell Culture Techniques; Cell Differentiation process; Cell Nucleus; Cell model; Cell physiology; Cells; Chemotaxis; Complex; Coronary; Coupled; Defect; Development; Differentiation Antigens; Disease; Ectopic Expression; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesions; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Growth; Growth Factor; Healed; In Vitro; Injury; Integrins; Investments; Knockout Mice; LIM Domain; Lasers; Life; Mediating; Membrane; Mesenchyme; Monitor; Monomeric GTP-Binding Proteins; Morphogenesis; Myocardial; Nuclear; Persistent Truncus Arteriosus; Phenotype; Play; Pluripotent Stem Cells; Process; Protein Tyrosine Kinase; Proteins; Pulmonary artery structure; Regulation; Role; Serum Response Factor; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Structure; Testing; Time; Tyrosine Phosphorylation; Vascular System; Yeasts; adapter protein; aortic arch; base; cell growth; cell motility; cell type; chromatin remodeling; fluorophore; gain of function; healing; homologous recombination; in vivo; in vivo Model; inhibitor/antagonist; injury and repair; interest; knock-down; leupaxin; loss of function; migration; myocardin; platelet-derived growth factor BB; promoter; public health relevance; reconstitution; repaired; research study; response; selective expression; trafficking; yeast two hybrid system

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.

描述（由申请人提供）：平滑肌细胞（SMC）生长、迁移和分化的精确调节对于血管的正常发育是必要的，这些过程的控制缺陷有助于几种突出的先天性和成人发作性心血管疾病的进展。广泛的研究表明，这些SMC的功能是由生长因子和细胞外基质（ECM）-整合素相互作用和激活的非受体酪氨酸激酶，粘着斑激酶（FAK）在这些信号通路中起着关键作用。我们以前已经证明，内源性抑制剂FAK，称为FRNK，选择性地表达在SMC中观察到特别高的水平在管道血管和FRNK表达显着上调在出生后的血管发育和血管损伤后。这些研究表明SMC中整合素基质信号传导是独特的，并且FAK活性的精确调节在血管发育和血管损伤修复中是至关重要的。事实上，我们已经证明，在wnt-1或nkx2.5衍生的SMC中FAK的条件性失活（通过同源重组）导致与出生后生活不相容的持续性动脉干。由于神经元分离涉及几个SMC过程的动态控制，我们继续研究FAK和FRNK在SMC中的作用，使用各种功能的损失/增益的方法。通过基因缺失抑制FAK活性对细胞生长或ERK活化几乎没有影响。然而，它强烈抑制PDGF-BB介导的细胞极化和迁移，这种作用可能是由于小GT3 Rac-1的激活缺陷。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性（通过SMC分化标志物基因表达进行评估）。 在试图进一步描绘参与这些反应的FAK依赖性机制，我们确定了LIM结构域衔接蛋白，leupaxin，在SMC中表达的FAK相互作用蛋白的酵母双杂交筛选。我们利用siRNA介导的方法从SMC中去除leupaxin，这些研究表明leupaxin对SMC的趋化性至关重要。我们还发现了一个有趣的和潜在的重要发现，亮帕素穿梭于粘着斑和细胞核之间，这一过程受到FAK信号的调节。我们还证明，leupaxin的异位表达上调多个SMC分化标志物基因; leupaxin的敲低减弱SMC分化标志物基因表达; leupaxin与SM α-肌动蛋白启动子在体内相互作用;以及leupaxin与SRF和强大的SRF辅因子myocardin在物理和功能上相互作用。 在这个建议中，我们试图确定FAK如何在血管形态发生过程中调节SMC表型，并确定FAK和leupaxin改变SMC运动和分化的确切机制。我们假设leupaxin用于整合这些不同的SMC功能，并通过靶向Rac-1激活运动细胞的前沿来调节迁移，并通过调节SMC分化标记基因启动子上功能性SRF转录因子复合物的形成来调节分化。