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，并在动脉损伤后在新内膜中积累。细胞周期进程受细胞周期蛋白/细胞周期蛋白依赖性激酶（cyclin/cyclin-dependent kinase， CDK）复合物的正调控，而受p21家族的负调控。P57kip2是p21家族成员之一，其分布相对局限于终末分化组织，包括肌肉。然而，p57kip2对VSMC增殖的贡献几乎是未知的。在这项研究中，我们研究了p57kip2与其他细胞周期相关蛋白联合在VSMC增殖中的作用。用血清剥夺法捕获VSMCs，然后用生长培养基刺激有丝分裂，每4小时收获一次。未成熟的VSMCs即使在有丝分裂剥夺后也没有分化。有丝分裂刺激导致p57kip2和p27kip1蛋白水平降低，与细胞周期蛋白- d、-E和CDK2蛋白量及其激酶活性上调相关，导致视网膜母细胞瘤（Rb）蛋白家族成员p107和p130磷酸化。免疫沉淀显示p27kip1可以与cyclin-D1结合，但不抑制其活性。相反，p57kip2可以结合cyclin-D1，并且p57kip2的过表达阻止了Rb家族的磷酸化，可能是由于取消了cyclin-D1（和/或cyclin-E）激酶的激活。综上所述，p57的下降可能是未成熟VSMCs增殖启动的关键事件。这些结果表明，p57kip2的补充过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.