Effect of CKI overexpression on Smooth Muscle Cell Growth

血管平滑肌细胞(VSMCs)在有丝分裂刺激下增殖增强，在动脉损伤后积聚在新生内膜中。细胞周期进程受细胞周期蛋白/细胞周期蛋白依赖性激酶(CDK)复合体的正向调控，受p21家族负向调控。P57Kip2是p21家族的成员之一，其分布相对局限于终末分化组织，包括肌肉。然而，p57Kip2在VSMC增殖中的作用仍不是未知的。在本研究中，我们结合其他细胞周期相关蛋白，研究了p57Kip2在VSMC增殖中的作用。用去血清的方法阻断VSMCs，然后用生长液刺激有丝分裂，每隔4h收集一次。未成熟VSMCs的表型即使在有丝分裂剥夺后也没有分化。有丝分裂刺激后p57kip2和p27kip1的蛋白水平降低，与细胞周期蛋白-D、-E和CDK2的蛋白含量及蛋白激酶活性上调有关，导致视网膜母细胞瘤蛋白家族成员p107和p130的磷酸化。免疫沉淀显示p27kip1可与细胞周期蛋白-D1结合，但不抑制其活性。相反，p57Kip2可与细胞周期蛋白-D1结合，并且过表达的p57Kip2可抑制Rb家族的磷酸化，这可能是通过取消细胞周期蛋白-D1(和/或细胞周期蛋白-E)的激活而实现的。综上所述，p57蛋白表达下降可能是未成熟VSMCs增殖启动的关键事件。这些结果提示，p57Kip2的过度表达可能是预防动脉损伤后内膜增生的有效策略。

Vascular smooth muscle cells (VSMCs) exhibit enhanced proliferation under mitogenic stimulation and accumulate in neointima after arterial injury. The cell cycle progression is positively regulated by cyclin/cyclin-dependent kinase (CDK) complex, and negatively controlled by p21 family. P57kip2 is one of the members of p21 family and its distribution is relatively limited to terminally differentiated tissues, including muscle. However, p57kip2 contribution to VSMC proliferation remains scarcely unknown. In this study, we investigated the role of p57kip2 in VSMC proliferation, in combination with other cell cycle related proteins. VSMCs were G0 arrested by serum deprivation, followed by mitogenic stimulation with growth medium, and harvested at every four hour. The phenotype of immature VSMCs didn't differentiate even after mitogenic deprivation. The protein levels of p57kip2 and p27kip1 diminished by mitogenic stimulation, in correlation with the upregulation of cyclin-D, -E and CDK2 protein amount and their kinase activities, resulting in the phosphorylation of retinoblastoma (Rb) protein family members, p107 and p130. Immunoprecipitation revealed that p27kip1 could associate with cyclin-D1, but did not inhibit its activity. On the contrary, p57kip2 could bind to cyclin-D1, and overexpressed p57kip2 prevented the phosphorylation of Rb family, probably due to canceling the cyclin-D1 (and/or cyclin-E) kinase activation. In conclusion, p57 decline is likely to be the key event for initiation of immature VSMCs proliferation. These results suggested that spplemental overexpression of p57kip2 could be a powerful strategy to prevent the development of intimal hyperplasia after arterial injury.