Control of Vascular Cell Motility by CaMKII

描述（由申请方提供）：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，并有助于血管疾病的进展。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，Ca 2 +/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CaMKII在这种情况下的激活机制，以及相关的底物靶点和对迁移的潜在影响机制尚不清楚。基于我们已发表的和初步的研究，我们假设，CaMK II 42的局部前沿激活调节局灶性粘附动力学，并作为一个积极的反馈，以促进VSM细胞极化和定向迁移。在目的1分子/遗传方法将被用来操纵CaMKII 42的表达和活性，并确定个别迁移VSM细胞的定向持久性和速度的后果。GFP标记的粘着斑成分的光漂白后荧光恢复（FRAP）分析将用于评估粘着斑动力学的CaMKII 4依赖性调节。在目的2生化和先进的荧光成像方法（共聚焦FRET和TIRF显微镜）将被用来测试一个潜在的机制，涉及功能性CaMKII 42与Src家族激酶，Fyn的相互作用，在调节粘着斑蛋白动力学。VSM细胞迁移是“合成表型”细胞的特性，其尚未获得或已丧失分化的收缩蛋白标记物和功能。我们已经确定CaMK II 4亚型（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，在合成表型VSM细胞中，TRPC和STIM/奥赖家族中某些Ca 2+传导阳离子通道的上调在功能上与增殖和迁移的调节相关。基于此，在目标3中，我们用分子和荧光成像方法来检验STIM 1/Orai 1介导的Ca 2+内流与前沿CaMKII激活功能相关的假设。在目标4中，我们测试的假设，即表达的CaMKII 4同工酶和这些Ca 2+通道的共同调节作为VSM表型的功能，赋予一个整合的途径，Ca 2+依赖性调节VSM细胞迁移在体内。这些新的研究结果，预计提供深入了解的机制，钙离子信号调节VSM细胞迁移，并提供了一个基础，了解钙离子依赖性调节迁移在其他细胞类型和过程，如肌纤维母细胞介导的伤口愈合或血管生成。与最近发表的心脏和骨骼肌的研究，这些研究表明，在VSM中CaMKII 4亚型可能是一般重要的肌肉重塑在生理或病理生理应激。 公共卫生相关性：通过阐明CaMKII亚型在血管损伤中的功能后果，这些研究有望为血管增生性疾病（包括动脉粥样硬化和血管手术后再狭窄）的潜在机制提供见解。由于Ca 2+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来自这些研究的信息可以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。

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.