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（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，并且这些过程的控制缺陷会导致几种突出的先天性和成人发病的心血管疾病的进展。大量研究表明，这些 SMC 功能受到生长因子和细胞外基质 (ECM)-整合素相互作用的调节，并且非受体酪氨酸激酶、粘着斑激酶 (FAK) 的激活在这些信号传导途径中发挥着关键作用。我们之前已经证明，FAK 的内源性抑制剂（称为 FRNK）在 SMC 中选择性表达，在导管血管中观察到水平特别高，并且 FRNK 表达在出生后血管发育和血管损伤后显着上调。这些研究表明 SMC 中的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过程中至关重要。事实上，我们已经证明，wnt-1 或 nkx2.5 衍生的 SMC 中 FAK 的条件失活（通过同源重组）会导致与产后生活不相容的持续性动脉干。由于主动脉肺动脉间隔涉及多个 SMC 过程的动态控制，因此我们使用各种功能丧失/增强方法继续研究 FAK 和 FRNK 在 SMC 中的作用。通过基因缺失抑制 FAK 活性对细胞生长或 ERK 激活几乎没有影响。然而，它强烈抑制 PDGF-BB 介导的细胞极化和迁移，这种效应可能是由于小 GTPase Rac-1 的激活缺陷所致。有趣的是，我们的体外和体内模型的结果也表明 FAK 活性与 SMC 分化之间存在很强的负相关性（通过 SMC 分化标记基因表达进行评估）。 为了进一步描述这些反应中涉及的 FAK 依赖性机制，我们在酵母双杂交筛选 SMC 中表达的 FAK 相互作用蛋白中鉴定了 LIM 结构域接头蛋白 leupaxin。我们利用 siRNA 介导的方法来消耗 SMC 中的 leupaxin，这些研究表明 leupaxin 对于 SMC 趋化性至关重要。我们还发现了一个有趣且可能重要的发现，即 leupaxin 在粘着斑和细胞核之间穿梭，并且该过程受到 FAK 信号传导的调节。我们还证明了 leupaxin 的异位表达上调了多个 SMC 分化标记基因； leupaxin 的敲除减弱了 SMC 分化标记基因的表达； leupaxin 在体内与 SM a-肌动蛋白启动子相互作用；并且 leupaxin 与 SRF 和强大的 SRF 辅助因子心肌素在物理和功能上相互作用。 在本提案中，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 运动和分化的精确机制。我们假设 leupaxin 可以整合这些不同的 SMC 功能，并通过将 Rac-1 激活靶向运动细胞的前缘来调节迁移，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。 公共健康相关性：平滑肌细胞的精确生长对于血管的正常形成和功能是必要的，并且在损伤后修复血管中也发挥着作用。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。本文提出的实验将结合使用细胞和遗传方法。