SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN VASCULAR SMOOTH MUSCLE CELL MIGRATION

血管平滑肌细胞迁移涉及的信号转导途径

• 批准号: 6431474
• 负责人: MICHAEL T CROW
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431474
• 关键词: Adenoviridae; biological signal transduction; calmodulin dependent protein kinase; cell migration; fibroblast growth factor; gene expression; integrins; platelet derived growth factor; transfection /expression vector; vascular smooth muscle

SUMMARY OF WORK Mechanical injury to blood vessels results in the relatively slow accumulation of vascular smothh muscle cells (VSMCs), which normally reside in the medial (M) cell layer, at the luminal interface to form a neointima (N), a process that mimics and is an experimental model for transplant atherosclerosis and vessel restenosis. We have previously shown that activation of calcium/calmodulin-dependent protein kinase II (CamKII) is a key intracellular signaling event regulating PDGF-directed vascular smooth muscle cell (VSMC) migration out of the media. CamKII integrates signals from integrins, such as alphav/beta3, integrated associated proteins (e.g., IAP),and extracellular matirx molecules, such as thrombospondin (TSP)to coordinate chemoattractant-induced cell movement into the neointima. More recently, we have investigated the role of apoptosis in neointima formation. Normal blood vessels do not express the muscle-specific repressor of apoptosis, known as ARC. However, ARC selectively accumulates in the developing neointima after vessel injury. N-VSMCs isolated from the site of injury show increased resistance to apoptosis compared to isolated M-VSMCs. Forced expression of ARC, but not its CARD-defective mutant, in M-VSMCs to levels comparable to that in N-VMSCs increases their resistance to apoptosis. Upregulation of ARC in respond to vessel injury, therefore, may confer on N-VSMCs a differntial resistant to apoptosis that contributes to neointimal cell accumulation. recognition. Accordingly, antibodies to TSP, IAP, or alphav/beta3 all block VSMC migration as well as CamKII activation. Forced expression of constitutively activated CamKII restores migration to antibody-treated cells as well as to cells isolated from IAP-/- mice. In other cell types, we have shown that outside-in signaling from the beta3 integrin complex to other integrins (integrin crosstalk) is also mediated by CamKII and its effects on myosin light chain kinase (MLCK), which in nonmuscle cells promotes stress fiber formation. In these cell types as well as in migrating VSMCs, pharmacological inhibitors of MLCK mimic the effect of CamKII activation, promote migration, and inhibit stress fiber formation while promoting cortical actin deposition. The inhibitory effect of CamKII on MLCK activity may be direct via phosphorylation of the enzyme or indirect through phosphorylation of intermediary molecules. One possible intermediary is TIAM, a rac-GEF that has recently been shown to be a substrate for CamKII phosphorylation. Phosphorylation of TIAM by CamKII results in increased rac activity, cortical actin formation, and MCLK inhibition through the rac-associated kinase, PAK. We have shown that a dominant negative mutant of TIAM inhibits PDGF-directed migration and this inhibition can be overriden by constitutuvely activated mutants of rac, but not of CamKII. These results demonstrate that the regulation of rac is a critical downstream effector of CamKII in the regulation of VSMC migration and the crosstalk between different integrins in the cell.

血管的机械损伤导致血管平滑肌细胞（VSMC）相对缓慢的积累，其通常位于中膜（M）细胞层中，在管腔界面处形成新生内膜（N），这一过程模拟并且是移植动脉粥样硬化和血管再狭窄的实验模型。我们以前已经表明，钙/钙调蛋白依赖性蛋白激酶II（CamKII）的激活是一个关键的细胞内信号事件调节PDGF指导的血管平滑肌细胞（VSMC）迁移出媒体。 CamKII整合来自整合素如α v/β 3的信号，整合相关蛋白（例如，IAP）和细胞外基质分子，如血小板反应蛋白（TSP），以协调趋化因子诱导的细胞运动进入新生内膜。最近，我们研究了细胞凋亡在新生内膜形成中的作用。正常血管不表达肌肉特异性细胞凋亡抑制因子，称为ARC。 然而，ARC选择性地积聚在血管损伤后的新生内膜中。 与分离的M-VSMC相比，从损伤部位分离的N-VSMC显示出增加的对凋亡的抗性。 在M-VSMCs中，ARC的强制表达，而不是其CARD缺陷突变体，与N-VMSC中的水平相当，增加了它们对凋亡的抵抗力。因此，血管损伤时ARC的上调可能赋予N-VSMCs对凋亡的不同抵抗力，从而导致新生内膜细胞积聚。识别.因此，针对TSP、IAP或α v/β 3的抗体都阻断VSMC迁移以及CamKII活化。 组成型激活的CamKII的强制表达恢复了向抗体处理的细胞以及从IAP-/-小鼠分离的细胞的迁移。 在其他类型的细胞中，我们已经表明，从β 3整合素复合物到其他整合素的由外向内的信号传导（整合素串扰）也是由CamKII及其对肌球蛋白轻链激酶（MLCK）的影响介导的，MLCK在非肌肉细胞中促进应力纤维形成。 在这些细胞类型以及迁移的VSMC中，MLCK的药理学抑制剂模拟CamKII活化的作用，促进迁移，并抑制应力纤维形成，同时促进皮质肌动蛋白沉积。 CamKII对MLCK活性的抑制作用可以是通过酶的磷酸化直接的或通过中间分子的磷酸化间接的。一种可能的中间体是TIAM，一种rac-GEF，最近已被证明是CamKII磷酸化的底物。 通过CamKII使TIAM磷酸化导致rac活性增加、皮质肌动蛋白形成以及通过rac相关激酶PAK抑制MCLK。 我们已经表明，TIAM的显性负突变体抑制PDGF定向迁移，这种抑制作用可以被组成性激活的外消旋突变体而不是CamKII的突变体所取代。 这些结果表明，在VSMC迁移和细胞中不同整合素之间的串扰的调节中，rac的调节是CamKII的关键下游效应物。