Control of Vascular Cell Motility by CaMKII

CaMKII 对血管细胞运动的控制

基本信息

批准号：
8235852
负责人：
HAROLD A SINGER
金额：
$ 39.11万
依托单位：
ALBANY MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-15 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8235852
关键词：
Atherosclerosis Biochemical Blood Vessels Calcium Calcium Channel Cations Cell physiology Cells Chemotaxis Complex Confocal Microscopy Contractile Proteins Data Development Disease Family Feedback Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Focal Adhesions Image Immigration In Vitro Individual Injury Isoenzymes Knowledge Link MAPK3 gene Mediating Mediator of activation protein Microscopy Modeling Molecular Genetics Muscle Myocardium Myofibroblast Operative Surgical Procedures Pathway interactions Phenotype Physiological Process Property Protein Dynamics Protein Isoforms Protein-Serine-Threonine Kinases Proteins Publishing Reagent Regulation Role STIM1 gene Signal Pathway Signal Transduction Skeletal Muscle Small Interfering RNA Smooth Muscle Myocytes Structure System Testing Tissues Tyrosine Phosphorylation Up-Regulation Vascular Diseases Vascular Smooth Muscle Wound Healing angiogenesis base calmodulin-dependent protein kinase II cell motility cell type fluorescence imaging in vivo insight interest migration molecular imaging neointima formation novel public health relevance response restenosis spatial relationship spatiotemporal src-Family Kinases stressor vascular smooth muscle cell migration vascular smooth muscle cell proliferation

项目摘要

DESCRIPTION (provided by applicant): Migration of vascular smooth muscle (VSM) cells occurs during development, wound healing, angiogenesis, and contributes to the progression of vascular disease. Published studies support a role for the ubiquitous multifunctional serine/threonine protein kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII in regulating VSM cell migration, but the mechanisms of CaMKII activation in this setting, and the relevant substrate targets and mechanisms underlying effects on migration, are unknown. Based on our published and preliminary studies we have hypothesized that localized leading edge activation of CaMKII42 regulates focal adhesion dynamics and acts as a positive feedback to promote VSM cell polarization and directional migration. In Aim 1 molecular/genetic approaches will be used to manipulate CaMKII42 expression and activity, and the consequences on directional persistence and velocity of individual migrating VSM cells determined. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-tagged focal adhesion components will be used to assess CaMKII4-dependent regulation of focal adhesion dynamics. In Aim 2 biochemical and advanced fluorescence imaging approaches (confocal FRET and TIRF microscopy) will be used to test a potential mechanism involving a functional CaMKII42 interaction with the Src-family kinase, Fyn, in regulating focal adhesion protein dynamics. VSM cell migration is a property of "synthetic phenotype" cells that have not acquired, or have lost, differentiated contractile protein markers and function. We have determined that up-regulation of a CaMKII4 isoform (42 or 4C) contributes to VSM cell synthetic phenotype functions (proliferation, migration). Recent studies indicate up-regulation of certain Ca2+ conducting cation channels in the TRPC and STIM/Orai families in synthetic phenotype VSM cells is linked functionally to regulation of proliferation and migration. Based on this, in Aim 3 we molecular and fluorescence imaging approaches to test the hypothesis that STIM1/Orai1 mediated Ca2+ entry is functionally linked to leading edge CaMKII activation. In aim 4 we test the hypothesis that that expression of CaMKII4 isozymes and these Ca2+ channels are co-regulated as a function of VSM phenotype, conferring an integrated pathway for Ca2+-dependent regulation of VSM cell migration in vivo. Results of these novel studies are expected to provide insight into mechanisms by which Ca2+ signals regulate VSM cell migration and to provide a basis for understanding Ca2+-dependent regulation of migration in other cell types and processes such as myofibroblast-mediated wound healing or angiogenesis. Taken with recently published studies in heart and skeletal muscle, these studies in VSM suggest that CaMKII4 isoforms may be generally important in muscle remodeling in response to physiological or pathophysiological stressors. PUBLIC HEALTH RELEVANCE: By elucidating the functional consequences of CaMKII isoforms in response to vascular injury, these studies are expected to provide insights into mechanisms underlying vascular proliferative diseases including atherosclerosis and restenosis follow vascular surgery. Because Ca2+ signals and CaMKII are a ubiquitous regulatory system in all cells and tissues, the information derived from these studies can be reasonably expected to extend to other processes involving vascular cell motility such as myofibroblast-mediated wound healing or angiogenesis.

描述（由申请人提供）：血管平滑肌（VSM）细胞的迁移发生在发育、伤口愈合、血管生成过程中，并导致血管疾病的进展。已发表的研究支持普遍存在的多功能丝氨酸/苏氨酸蛋白激酶、Ca2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 在调节 VSM 细胞迁移中的作用，但在这种情况下 CaMKII 激活的机制以及相关底物靶标和潜在作用机制根据我们已发表的初步研究，我们假设 CaMKII42 的局部前缘激活可调节粘着斑动力学，并作为促进 VSM 细胞极化的正反馈。在目标 1 中，将使用分子/遗传方法来操纵 CaMKII42 表达和活性，并确定对 GFP 标记的粘着斑进行光漂白 (FRAP) 分析后对单个迁移 VSM 细胞的定向持久性和速度的影响。 In Aim 2 生物化学和先进荧光成像方法（共焦 FRET 和 TIRF 显微镜）将用于测试粘着斑动力学的 CaMKII4 依赖性调节。潜在机制涉及 CaMKII42 与 Src 家族激酶 Fyn 的功能性相互作用，调节粘着斑蛋白动力学。 VSM 细胞迁移是“合成表型”细胞的一个特性，这些细胞尚未获得或丢失分化的收缩蛋白标记和功能。我们已经确定 CaMKII4 同工型（42 或 4C）的上调有助于 VSM 细胞合成表型功能（增殖、迁移）。最近的研究表明，合成表型 VSM 细胞中 TRPC 和 STIM/Orai 家族中某些 Ca2+ 传导阳离子通道的上调与增殖和迁移的调节在功能上相关。基于此，在目标 3 中，我们采用分子和荧光成像方法来检验 STIM1/Orai1 介导的 Ca2+ 进入与前沿 CaMKII 激活在功能上相关的假设。在目标 4 中，我们测试了以下假设：CaMKII4 同工酶和这些 Ca2+ 通道的表达作为 VSM 表型的函数进行共同调节，从而为 VSM 细胞迁移的体内 Ca2+ 依赖性调节提供了一条整合途径。这些新研究的结果有望深入了解 Ca2+ 信号调节 VSM 细胞迁移的机制，并为理解其他细胞类型和过程（如肌成纤维细胞介导的伤口愈合或血管生成）中 Ca2+ 依赖性迁移调节提供基础。结合最近发表的心脏和骨骼肌研究，VSM 中的这些研究表明，CaMKII4 同工型可能在响应生理或病理生理应激源的肌肉重塑中通常很重要。公共健康相关性：通过阐明 CaMKII 同工型对血管损伤的功能影响，这些研究有望提供对血管增殖性疾病（包括动脉粥样硬化和血管手术后再狭窄）的潜在机制的见解。由于 Ca2+ 信号和 CaMKII 是所有细胞和组织中普遍存在的调节系统，因此可以合理地预期从这些研究中获得的信息将扩展到涉及血管细胞运动的其他过程，例如肌成纤维细胞介导的伤口愈合或血管生成。