SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN VASCULAR SMOOTH MUSCLE CELL MIGRATION

我们先前表明，钙/钙调蛋白依赖性蛋白激酶II（CamKII）的激活是调节PDGF指导的血管平滑肌细胞（VSMC）迁移的关键细胞内信号传导事件。我们目前的工作集中在α v/β 3整合素复合物在CamKII依赖性信号传导中的作用。在血管平滑肌细胞中，α v/β 3与整合素相关蛋白（IAP）形成复合物，形成独特的受体复合物，用于识别血小板反应蛋白（TSP）。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。- 迁移、平滑肌、细胞外基质、整合素、钙、血小板反应蛋白、肌球蛋白、动脉硬化、再狭窄

SUMMARY OF WORK We previously showed 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. Our current work is focused on the role of the alphav/beta3 integrin complex in CamKII-dependent signaling. In VSMCs, alphav/beta3 exists in a complex with integrin-assocaited protein (IAP), creating a unique receptor complex for thrombospondin (TSP) 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. - Migration, Smooth muscle, extracellular matrix, integrins, calcium, thrombospondin, myosin, arteriosclerosis, restenosis