Regulation of proliferatin in vascular endothelial and smooth muscle cells by the protein kinase C pathway

为探讨蛋白激酶C(PKC)在血管细胞增殖中的作用，我们观察了佛波酯和1-油酰基-2-乙酰甘油(OAG)对血管内皮细胞和平滑肌细胞周期的影响，并以G0同步化的人脐动脉平滑肌细胞为模型，研究了PKC在G1/S细胞周期中的作用。用20%胎牛血清和10 ng/ml碱性成纤维细胞生长因子刺激15h后，胸腺嘧啶核苷开始掺入。PMA在3h前加入可抑制90%以上的掺入，6h或以后加入PMA则抑制作用减弱。PMA抑制视网膜母细胞瘤蛋白(PRB)的磷酸化，通常在9h开始。PMA抑制CDK2的活性，从9h开始升高，而PMA不抑制CDK4/6的活性，从0-3h开始增加。从3h重复加入OAG(10muM)也抑制[~3H]胸腺嘧啶核苷的掺入，Prb的磷酸化和CDK2作用的…更有趣的是。PMA对细胞周期蛋白CDK2、CDK3、CDK4、CDK5和细胞周期蛋白G、C、D的基因表达无抑制作用，而对细胞周期蛋白E和A的基因表达有抑制作用，它们分别从3~9h和15h开始。但PMA不降低细胞周期蛋白E和A的蛋白水平，对CDK抑制剂p21和p27的表达无影响。在SDS-PAGE中，PMA抑制CDK2向代表Thr160磷酸化和Tyr15去磷酸化的较慢迁移形式的转变。PMA引起G2期细胞停滞的原因是：第一，当PMA加入到G2细胞中时，抑制了随后的细胞分裂；第二，这些生长停滞的细胞没有表现出有丝分裂细胞的形态特征；第三，PMA没有中断诺考达唑诱导的M期停滞释放的细胞的有丝分裂。OAG还能抑制微丝分裂。PMA和OAG抑制了CDc2蛋白在G_2/M期的活化，但PMA抑制了其酪氨酸残基的去磷酸化。同时，PMA抑制了CDc25B的表达。PMA减少了细胞周期蛋白B的总量和与CDc2相关的量。因此，PKC通路分别通过抑制CDK2和CDC2来负向调节细胞的G_1/S和G_2/M期转换。CDK活性的抑制可能是由于抑制了苏氨酸磷酸化、酪氨酸去磷酸化以及它们的伴侣细胞周期蛋白的表达。较少

To elucidate the role of protein kinase C (PKC) in vascular cell proliferation, we examined the effects of phorbol-myristate-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) on the cell cycle events in smooth muscle and endothelial cells.The role of PKC in the G_1/S transition was studied using G_0-synchronized human umbilical artery smooth muscle cells. [^3H] thymidine incorporation started 15 h after stimulation with 20% fetal bovine serum and 10ng/ml basic fibroblast growth factor. PMA inhibited the incorporation over 90% when added earlier than 3 h, but the inhibition was attenuated when PMA was added at 6 h or later. PMA inhibited the phosphorylation of the retinoblastoma rotein (pRb), which normally began at about 9 h. PMA inhibited the activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit Cdk4/6 activities, which increased from 0-3 h. OAG (10muM) added repeatedly from 3 h also inhivited [^3H] thymidine incorporation, pRb phosphorylation, and Cdk2 act … More ivity. PMA did not inhibit the mRNA expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G,C,and D,all of which began at 0-3 h, whereas PMA reduced the mRNA expression of cyclins E and A,which usually began at 3-9 h and about 15 h, respectively. However, PMA did not reduce the protein levels of cyclins E and A.PMA had no influence on the expression of Cdk inhibitors p21 and p27. PMA inhibited the shift of Cdk2 to a slower migrating form in SDS-PAGE that represents Thr160 phosphorylation and Tyr15 dephosphorylation.The role of PKC in the G_2/M transition was investigated in human umbilical vein endothelial cells released from the G_1/S border. PMA caused G_2 arrest because, firstly, when added to G_2 cells, PMA inhibited subsequent cell division, secondly, these growth-arrested cells did not show morphological features of mitotic cells, and thirdly, PMA did not interrupt mitosis in cells released from nocodazole-induced M phase arrest. OAG also inhibited moitosis. The activation of Cdc2 kinase around the G_2/M transition was suppressed by PMA and OAG.Although Cdc2 was expressed in the presence of PMA,dephosphorylation of its tyrosine residue was inhibited by PMA.In parallel, the expression of Cdc25B was suppressed by PMA.The total and the Cdc2 associated amount of cyclin B were both reduced by PMA.Therefore the PKC pathway negatively regulates both the G_1/S and G_2/M transitions by inhibiting Cdk2 and Cdc2, respectively. The suppression of Cdk activities may result from inhibited threonine phosphorylation, tyrosine dephosphorylation, and expression of their partner cyclins. Less