Rac1 in Smooth Muscle

平滑肌中的 Rac1

• 批准号: 8505529
• 负责人: Mitsuo Ikebe
• 金额: $ 43.22万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-05 至 2013-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505529
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Address; Adhesions; Agonist; Arteries; Asthma; Binding; Biochemical; Biological; Blood Pressure; Blood Vessels; Cell membrane; Cells; Cytoskeletal Modeling; Cytoskeleton; DNA Sequence Rearrangement; Disease; Dominant-Negative Mutation; Down-Regulation; Electron Microscopy; Extracellular Matrix; Filament; Fluorescence; Fluorescent Antibody Technique; Gene Silencing; Goals; Hypertension; Image; Laboratories; Link; Measures; Mechanics; Membrane; Microfilaments; Microscope; Molecular; Monitor; Movement; Muscle Contraction; Myosin ATPase; Myosin Light Chains; Organ; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein phosphatase; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Relaxation; Research; Resolution; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Stream; Structure; Techniques; Tissues; base; constriction; digital; enzyme activity; fluorescence microscope; genetic regulatory protein; improved; inhibitor/antagonist; insight; myosin phosphatase; novel; polymerization; tomography; tool; transmission process; two-photon

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the novel regulatory mechanism by which the Rac1 pathway regulates smooth muscle contraction. The smooth muscle contraction is a critical component for the regulation of constriction of hollow organs such as airway and arteries, thus controlling airflow and blood pressure, therefore, the proposed study will provide a novel insight into vascular and airway diseases. Smooth muscle contraction is primarily regulated by myosin light chain (MLC) phosphorylation, however, recent studies have suggested that actin cytoskeletal rearrangement may be in part responsible for the change in contraction. In this proposal, we hypothesize that the Rac signaling pathway concertedly controls smooth muscle contraction by changing MLC phosphorylation and cytoskeletal rearrangement. MLC phosphorylation is regulated by both Ca2+ dependent and Ca2+ independent pathways, and MLC phosphatase (MLCP) plays a key role in the latter mechanism. MLCP activity is regulated by the phosphorylation of MYPT1, a myosin binding regulatory subunit of MLCP, and CPI-17, a MLCP specific inhibitor. The research in the past has focused on the kinases responsible for MYPT1 and CPI-17 phosphorylation, such as Rho kinase and PKC, but nothing is known about the protein phosphatases that dephosphorylate MYPT1 and CPI-17. Based upon our findings, we hypothesize that the Rac pathway regulates MYPT1/CPI-17 phosphatases during agonist stimulation, which regulates MLCP and is in part responsible for the Rac dependent contractile regulation. Since smooth muscle undergoes rapid mechanical plasticity involving actin cytoskeletal change, we hypothesize that agonist stimulation induces Rac translocation to the membrane, where it activates its down-stream targets such as WAVE and PAK to recruit adhesion junction proteins, which strengthen the connections between the membrane adhesion junctions and actomyosin filaments to transmit force. We will first determine if Rac1 is activated after agonist stimulation. To evaluate the role of Rac1 in contraction, we will use pharmacological specific Rac inhibitors and molecular biological tools and gene silencing. Furthermore, we will clarify the mechanism by which Rac activation regulates the contraction. The change in MYPT1 phosphatase and/or CPI-17 phosphatase activities will be determined along with the Rac activity change using biochemical means. We will also examine if Rac1 activation stimulates the actin cytoskeletal change via WAVE and ARP2/3 translocation to the cell periphery. We will measure actin polymerization, and the binding of Rac and its down stream proteins. Translocation of Rac1 and its down-stream targets will also be studied with arterial tissues and single cells using a two-photon digital microscope, 3D digital confocal microscope, and a total internal reflection fluorescence (TIRF) microscope with super resolution analysis. The Rac1 dependent ultrastructural change will be achieved by electron microscopy using tomography technique to obtain 3D structural images.

描述（由申请人提供）：该项目的目标是阐明 Rac1 通路调节平滑肌收缩的新调节机制。平滑肌收缩是调节气道和动脉等中空器官收缩的关键组成部分，从而控制气流和血压，因此，这项研究将为血管和气道疾病提供新的见解。平滑肌收缩主要由肌球蛋白轻链 (MLC) 磷酸化调节，然而，最近的研究表明肌动蛋白细胞骨架重排可能是收缩变化的部分原因。在这个提议中，我们假设 Rac 信号通路通过改变 MLC 磷酸化和细胞骨架重排来协调控制平滑肌收缩。 MLC 磷酸化受 Ca2+ 依赖性和 Ca2+ 非依赖性途径调节，MLC 磷酸酶 (MLCP) 在后一种机制中发挥关键作用。 MLCP 活性由 MYPT1（MLCP 的肌球蛋白结合调节亚基）和 CPI-17（MLCP 特异性抑制剂）的磷酸化调节。过去的研究主要集中在负责MYPT1和CPI-17磷酸化的激酶上，例如Rho激酶和PKC，但对使MYPT1和CPI-17去磷酸化的蛋白磷酸酶一无所知。根据我们的发现，我们假设 Rac 通路在激动剂刺激期间调节 MYPT1/CPI-17 磷酸酶，从而调节 MLCP，并部分负责 Rac 依赖性收缩调节。由于平滑肌经历涉及肌动蛋白细胞骨架变化的快速机械可塑性，我们假设激动剂刺激诱导 Rac 易位至膜，激活其下游靶标（如 WAVE 和 PAK）以招募粘附连接蛋白，从而加强膜之间的连接粘附连接和肌动球蛋白丝传递力。我们首先确定 Rac1 在激动剂刺激后是否被激活。评估角色 为了抑制Rac1的收缩，我们将使用药理特异性Rac抑制剂和分子生物学工具以及基因沉默。此外，我们将阐明 Rac 激活调节收缩的机制。将使用生化方法测定 MYPT1 磷酸酶和/或 CPI-17 磷酸酶活性的变化以及 Rac 活性的变化。我们还将检查 Rac1 激活是否通过 WAVE 和 ARP2/3 易位至细胞外周刺激肌动蛋白细胞骨架的变化。我们将测量肌动蛋白聚合以及 Rac 及其下游蛋白的结合。还将使用双光子数字显微镜、3D 数字共聚焦显微镜和具有超分辨率分析功能的全内反射荧光 (TIRF) 显微镜，通过动脉组织和单细胞研究 Rac1 及其下游靶标的易位。 Rac1 依赖性超微结构变化将通过电子显微镜使用断层扫描技术获得 3D 结构图像来实现。