Control of Vascular Cell Motility by CaMKII

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，Ca2+/钙调素依赖性蛋白激酶II （CaMKII）在调节VSM细胞迁移中的作用，但CaMKII在这种情况下激活的机制，以及相关的底物靶点和迁移影响的机制尚不清楚。基于我们已发表的和初步的研究，我们假设CaMKII42的局部前缘激活调节了局部粘附动力学，并作为正反馈促进了VSM细胞的极化和定向迁移。在Aim 1中，分子/遗传方法将用于操纵CaMKII42的表达和活性，并确定个体迁移VSM细胞的定向持久性和速度的后果。对gfp标记的黏附组分进行光漂白后荧光恢复（FRAP）分析，以评估camkii4依赖性的黏附动力学调节。在Aim 2中，生化和先进的荧光成像方法（共聚焦FRET和TIRF显微镜）将用于测试CaMKII42与src家族激酶Fyn的功能相互作用在调节黏附蛋白动力学中的潜在机制。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Ca2+传导阳离子通道的上调在功能上与增殖和迁移的调节有关。基于此，在Aim 3中，我们采用分子和荧光成像方法来验证STIM1/Orai1介导的Ca2+进入与前沿CaMKII激活在功能上相关的假设。在目的4中，我们验证了CaMKII4同工酶的表达和这些Ca2+通道作为VSM表型的功能共同调节的假设，从而为VSM细胞在体内迁移的Ca2+依赖性调节提供了一个综合途径。这些新研究的结果有望提供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+依赖的迁移调节提供基础。结合最近发表的心脏和骨骼肌的研究，这些在VSM上的研究表明，CaMKII4亚型可能在响应生理或病理生理应激源的肌肉重塑中普遍重要。

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.