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

冠状动脉和流出道脱皮过程中粘附依赖性 SMC 可塑性的控制

• 批准号: 8039991
• 负责人: Joan M Taylor
• 金额: $ 33.3万
• 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8039991
• 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 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. 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.

描述（申请人提供）：精确调节平滑肌细胞（SMC）的生长、迁移和分化是血管正常发育所必需的，这些过程的控制缺陷导致了一些突出的先天性和成年性心血管疾病的进展。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，Focal Adhesion kinase （FAK）的激活在这些信号通路中起着关键作用。我们之前已经证明，FAK的内源性抑制剂FRNK在SMC中选择性表达，在导管血管中观察到特别高的水平，并且FRNK的表达在出生后血管发育和血管损伤期间显着上调。这些研究表明SMC中的整合素基质信号是独特的，FAK活性的精确调控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衍生的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动脉干。由于主动脉肺分离涉及多个SMC过程的动态控制，我们继续使用各种功能损失/增益方法研究FAK和FRNK在SMC中的作用。基因缺失对FAK活性的抑制对细胞生长和ERK激活的影响不大。然而，它强烈抑制pdgf - bb介导的细胞极化和迁移，这可能是由于小GTPase Rac-1激活缺陷所致。有趣的是，我们体外和体内模型的结果也表明FAK活性与SMC分化之间存在很强的负相关（通过SMC分化标记基因表达来评估）。为了进一步描述参与这些反应的FAK依赖机制，我们在酵母双杂交筛选中发现了LIM结构域适配蛋白，leupaxin，用于在SMC中表达的FAK相互作用蛋白。我们利用sirna介导的方法从SMC中消耗白紫色素，这些研究表明白紫色素对SMC趋化性至关重要。我们还发现了一个有趣且潜在重要的发现，即leupaxin在局灶黏着物和细胞核之间穿梭，这一过程是由FAK信号传导调节的。我们还证明了异位表达leupaxin上调了多个SMC分化标记基因；leupaxin的敲除降低了SMC分化标记基因的表达；leupaxin在体内与SM - a-actin启动子相互作用；leupaxin与SRF和强大的SRF辅助因子心肌素在生理和功能上相互作用。在本研究中，我们试图确定FAK在血管形态发生过程中如何调节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整合了这些不同的SMC功能，并通过将Rac-1激活到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。