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

蛋白激酶 C 通路对血管内皮细胞和平滑肌细胞增殖的调节

• 批准号: 07833014
• 负责人: SASAGURI Toshiyuki
• 金额: $ 1.54万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07833014/
• 关键词: Protein kinase C; Vascular endothelial cells; Vascular smooth muscle cells; Cell proliferation; Cell cycle

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

为了阐明蛋白激酶C （PKC）在血管细胞增殖中的作用，我们研究了磷酸肉豆蔻酸酯（PMA）和1-油基-2-乙酰基- asn -甘油（OAG）对平滑肌和内皮细胞细胞周期事件的影响。利用与G_1/S同步的人脐动脉平滑肌细胞研究PKC在G_1/S转变中的作用。[^3H]在20%胎牛血清和10ng/ml碱性成纤维细胞生长因子刺激后15h开始加入胸苷。PMA加入时间早于3 h时，抑制作用超过90%，但加入时间晚于6 h时，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9 h左右开始。PMA抑制Cdk2的活性，从9 h开始增加，而PMA不抑制Cdk4/6的活性，从0-3 h开始增加。OAG （10muM）从3 h开始重复添加也抑制[^3H]胸苷结合，pRb磷酸化和Cdk2的活性。PMA不抑制Cdk2、Cdk3、Cdk4、Cdk5和细胞周期蛋白G、C、D的mRNA表达，它们都在0-3 h开始表达，而PMA降低了细胞周期蛋白E和A的mRNA表达，它们通常分别在3-9 h和15 h左右开始表达。然而，PMA并没有降低细胞周期蛋白E和a的蛋白水平，PMA对Cdk抑制剂p21和p27的表达没有影响。PMA抑制Cdk2在SDS-PAGE中向较慢的迁移形式转移，这代表Thr160磷酸化和Tyr15去磷酸化。研究了PKC在G_1/S边界释放的人脐静脉内皮细胞向G_2/M过渡中的作用。PMA引起G_2期阻滞的原因有三：首先，加入G_2期阻滞细胞后，PMA抑制了随后的细胞分裂；其次，这些生长阻滞细胞不表现出有丝分裂细胞的形态特征；第三，在诺可达唑诱导的M期阻滞释放的细胞中，PMA不中断有丝分裂。OAG还能抑制潮湿。PMA和OAG抑制了Cdc2激酶在G_2/M转变周围的活化。尽管Cdc2在PMA存在下表达，但PMA抑制了其酪氨酸残基的去磷酸化。同时，PMA抑制Cdc25B的表达。PMA降低了细胞周期蛋白B的总量和Cdc2相关量。因此，PKC通路通过抑制Cdk2和Cdc2分别负向调节G_1/S和G_2/M的转变。Cdk活性的抑制可能是由于抑制苏氨酸磷酸化、酪氨酸去磷酸化及其伴侣细胞周期蛋白的表达。少

项目成果

Chiya Kosaka et al: "Cell cycle arrest in the G_2 phase induced by phorbol ester and diacylglycerol in vascular endothelial cells" Am.J.Physiol.270. C170-C178 (1996)

Chiya Kosaka 等人：“血管内皮细胞中佛波酯和二酰甘油诱导的细胞周期停滞在 G_2 期”Am.J.Physiol.270。

Toshiyuki Sasaguri et al.: "Phorbol ester inhibits the phosphorylation of the retinoblastoma protein without suppressing cyclin D-associated kinase in vascular smooth muscle cells." Journal of Biological Chemistry. 271. 8345-8351 (1996)

Toshiyuki Sasaguri 等人：“佛波酯抑制视网膜母细胞瘤蛋白的磷酸化，而不抑制血管平滑肌细胞中的细胞周期蛋白 D 相关激酶。”

Chiya Kosaka: "Cell cycle arrest in the G_2 phase induced by phorbol ester and diacylglycerol in vascular endothelial cells." Am. J. Physiol.270. C170-C178 (1996)

Chiya Kosaka：“佛波酯和二酰甘油在血管内皮细胞中诱导细胞周期停滞在 G_2 期。”

Sasaguri, T., Ishida, A., Kosaka, C., Nojima, H., and Ogata, J.: "Phorbol ester inhibits the phosphorylation of the retinoblastoma protein without suppressing cyclin D-associated kinase in vascular smooth muscle cells." J.Biol.Chem.271. 8345-8351 (1996)

Sasaguri, T.、Ishida, A.、Kosaka, C.、Nojima, H. 和 Ogata, J.：“佛波酯抑制视网膜母细胞瘤蛋白的磷酸化，而不抑制血管平滑肌细胞中的细胞周期蛋白 D 相关激酶。”

Kosaka, C., Sasaguri, T., Zen, K., Masuda, J., Shimokado, K., and Ogata, J.: "The protein kinase C pathway inhibits the proliferation of cultured vascular endothelial cells reducing cyclin A gene expression." Ann.N.Y.Acad.Sci.748. 538-540 (1995)

Kosaka, C.、Sasaguri, T.、Zen, K.、Masuda, J.、Shimokado, K. 和 Ogata, J.：“蛋白激酶 C 途径抑制培养的血管内皮细胞的增殖，减少细胞周期蛋白 A 基因的表达