Rac1 in Smooth Muscle

平滑肌中的 Rac1

• 批准号: 8373480
• 负责人: Mitsuo Ikebe
• 金额: $ 46.87万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8373480
• 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. PUBLIC HEALTH RELEVANCE: Smooth muscle plays an important role in the constriction/dilation of hollow organs such as airway and blood vessels and controls airway flow and blood pressure. Therefore, its malfunction causes various diseases such as high blood pressure and asthma. The proposed project will clarify the role of the novel regulatory pathway, i.e., the Rac signaling pathway, linking the external stimuli and the contraction of smooth muscle, and thus will improve our understanding of vascular and airway diseases.

描述(由申请者提供)：本项目的目标是阐明rac1通路调节平滑肌收缩的新的调节机制。平滑肌收缩是调节气道、动脉等中空器官收缩的重要组成部分，从而控制气流和血压，因此，本研究将为血管和呼吸道疾病提供新的视角。平滑肌收缩主要受肌球蛋白轻链(MLC)磷酸化的调节，然而，最近的研究表明，肌动蛋白细胞骨架重排可能是收缩改变的部分原因。在这个方案中，我们假设RAC信号通路通过改变MLC的磷酸化和细胞骨架重排来协调地控制平滑肌的收缩。MLC的磷酸化受钙依赖和非钙依赖两种途径的调节，而MLC磷酸酶(MLCP)在后一种机制中起关键作用。MLCP的活性由MLCP的肌球蛋白结合调节亚基MYPT1和MLCP特异性抑制物CPI-17的磷酸化调节。过去的研究主要集中在参与MYPT1和CPI-17磷酸化的激酶，如Rho激酶和PKC，但对使MYPT1和CPI-17去磷酸化的蛋白磷酸酶知之甚少。根据我们的发现，我们假设在激动剂刺激过程中，RAC通路调节MYPT1/CPI-17磷酸酶，从而调节MLCP，并部分地参与RAC依赖的收缩调节。由于平滑肌经历了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移位到膜上，在那里它激活其下游靶标如WAVE和PAK来招募黏附连接蛋白，从而加强膜黏附连接和肌动球蛋白细丝之间的联系以传递力量。我们将首先确定在激动剂刺激后，rac1是否被激活。评估角色的步骤 对于处于收缩状态的rac1，我们将使用药理学特异性的RAC抑制剂和分子生物学工具和基因沉默。此外，我们还将阐明Rac激活调节收缩的机制。MYPT1磷酸酶和/或CPI-17磷酸酶活性的变化将与RAC活性的变化一起使用生化手段来确定。我们还将研究rac1激活是否通过WAVE和Arp2/3易位到细胞外围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的全内反射荧光(TIRF)显微镜，在动脉组织和单细胞中研究rac1及其下游靶的移位。使用断层扫描技术获得三维结构图像的电子显微镜将实现rac1依赖的超微结构变化。 公共卫生相关性：平滑肌在呼吸道和血管等中空器官的收缩/扩张中发挥重要作用，并控制呼吸道流量和血压。因此，它的故障会导致高血压和哮喘等各种疾病。该项目将阐明连接外界刺激和平滑肌收缩的新的调控途径，即RAC信号通路的作用，从而提高我们对血管和呼吸道疾病的理解。