Control of Vascular Cell Motility by CaMKII

描述(由申请人提供)：血管平滑肌(VSM)细胞在发育、伤口愈合、血管生成过程中发生迁移，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，钙/钙调素依赖的蛋白激酶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，以及相关的底物靶点和潜在的影响迁移的机制，尚不清楚。根据我们已发表的和初步的研究，我们假设CaMKII42的局部前沿激活调节焦点黏附动力学，并作为正反馈促进VSM细胞极化和定向迁移。在目标1中，将使用分子/遗传学方法来调控CaMKII42的表达和活性，并确定对单个VSM细胞定向持续和迁移速度的影响。对GFP标记的焦点黏附成分的荧光漂白后恢复(FRAP)分析将用于评估CaMKII4依赖的焦点黏附动态调节。在AIM中，将使用两种生化和先进的荧光成像方法(共聚焦FRET和TIRF显微镜)来测试CaMKII42与Src家族激酶Fyn在调节焦点黏附蛋白动力学中的功能相互作用的潜在机制。VSM细胞迁移是“合成表型”细胞的一种特性，这些细胞没有获得或失去分化的收缩蛋白标记物和功能。我们已经确定CaMKII4亚型(42或4C)的上调有助于VSM细胞的合成表型功能(增殖、迁移)。最近的研究表明，在合成表型VSM细胞中，TRPC和STIM/Orai家族中某些钙离子传导阳离子通道的上调与增殖和迁移的调控有关。基于此，在目标3中，我们用分子和荧光成像方法验证了STIM1/Orai1介导的钙内流在功能上与前沿CaMKII激活有关的假说。在目标4中，我们验证了CaMKII4同工酶和这些钙通道表达作为VSM表型的函数而共同调节的假设，为体内VSM细胞迁移的钙依赖调节提供了一条完整的途径。这些新的研究结果有望为深入了解钙信号调节血管内皮细胞迁移的机制提供依据，并为理解其他细胞类型和过程中钙依赖的迁移调控提供基础，如肌成纤维细胞介导的伤口愈合或血管生成。结合最近发表的心脏和骨骼肌研究，VSM的这些研究表明，CaMKII4亚型可能在响应生理或病理生理应激的肌肉重塑中通常起重要作用。 公共卫生相关性：通过阐明CaMKII亚型在应对血管损伤时的功能后果，这些研究有望为血管手术后动脉粥样硬化和再狭窄等血管增生性疾病的潜在机制提供见解。由于钙信号和CaMKII是所有细胞和组织中普遍存在的调节系统，从这些研究中获得的信息可以合理地扩展到其他涉及血管细胞运动的过程，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.