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，我们用G_0期同步化的人脐动脉平滑肌细胞研究了佛波酯（PMA）和1-油酰基-2-乙酰基-sn-甘油（OAG）对血管平滑肌细胞和内皮细胞周期事件的影响。用20%胎牛血清和10 ng/ml碱性成纤维细胞生长因子刺激后15 h开始[^3H]胸苷掺入。PMA在3 h前加入可抑制90%以上的掺入，而在6 h或6 h后加入则抑制作用减弱。PMA抑制视网膜母细胞瘤蛋白（pRb）的磷酸化，这通常在约9小时开始。PMA抑制Cdk 2活性，Cdk 2活性从9 h开始升高，而PMA不抑制Cdk 4/6活性，Cdk 4/6活性从0-3 h开始升高。从3小时开始反复加入OAG（10 μ M）也抑制了[^3H]胸苷掺入、pRb磷酸化和Cdk 2激活。 关于我们 生命力PMA不抑制Cdk 2、Cdk 3、Cdk 4、Cdk 5和细胞周期蛋白G、C和D的mRNA表达，均在0-3 h开始，而PMA降低细胞周期蛋白E和A的mRNA表达，通常分别在3-9 h和15 h左右开始。PMA对Cdk抑制剂p21和p27的表达无影响。PMA可抑制Cdk 2的迁移，使Cdk 2在SDS-PAGE上表现为Thr 160磷酸化和Tyr 15去磷酸化。PMA引起G_2期阻滞的原因是：（1）当加入到G_2期细胞中时，PMA抑制了随后的细胞分裂;（2）这些生长阻滞的细胞不具有有丝分裂细胞的形态特征; OAG也抑制有丝分裂。PMA和OAG均能抑制Cdc 2激酶在G_2/M期的活化，但PMA能抑制Cdc 2激酶酪氨酸残基的去磷酸化，而PMA能抑制Cdc 2激酶在G_2/M期的活化。PMA可抑制Cdc 25 B的表达，使细胞周期蛋白B的总量和Cdc 2结合量均减少，从而使PKC途径负性调节G_1/S期和G_2/S期细胞周期。通过抑制Cdk 2和Cdc 2，M跃迁。Cdk活性的抑制可能是由于抑制了苏氨酸磷酸化、酪氨酸去磷酸化和它们的伴侣细胞周期蛋白的表达。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