Sasaguri T, Kosaka C, Hirata M, Masuda J, Shimokado K, Fujishima M, Ogata J
National Cardiovascular Center Research Institute, Osaka, Japan.
Exp Cell Res. 1993 Sep;208(1):311-20. doi: 10.1006/excr.1993.1251.
The role of protein kinase C (PKC) in the regulation of vascular smooth muscle cell proliferation was studied using not only phorbol ester but also diacylglycerol, with regard to the molecular species of PKC. Phorbol 12,13-dibutyrate (PDBu) and 1,2-dioctanoylglycerol (DOG) both potently inhibited serum-stimulated DNA synthesis and cell population doubling. The PDBu effect on DNA synthesis was maximal when applied at late G1. Neither PDBu nor DOG inhibited DNA synthesis in cells incubated with phorbol 12-myristate 13-acetate (PMA) for 24 h, which down-regulates PKC. Moreover, long exposure to PMA shortened the G1 period and the cell population doubling time. Therefore, a PKC isoform(s) that can be activated by phorbol ester and down-regulated by long exposure to PMA should be involved in the G1/S inhibition. A PKC enzyme assay of the soluble proteins extracted from late G1 cells and fractionated by anion exchange and hydroxylapatite chromatography showed that the activity eluting with PKC-alpha predominated, whereas that eluting with PKC-zeta was detectable. The former was dependent on Ca2+ and phorbol ester but the latter was not. PKC-zeta appeared to be expressed as two subspecies of M(r) 70 and 80 kDa. In cells incubated with PMA for 24 h, the activity eluting with PKC-alpha was completely abolished, whereas the significant activity eluting with PKC-zeta (70 kDa) remained. On the other hand, a relatively low, Ca(2+)-independent activity eluted with PKC-epsilon from the particulate fraction. This was reduced by long exposure to PMA, although not completely. Therefore, PKC-alpha and -epsilon may be the most probable mediators of the G1/S inhibition.
利用佛波酯和二酰甘油，从蛋白激酶C（PKC）的分子种类方面研究了PKC在调节血管平滑肌细胞增殖中的作用。佛波醇12,13 - 二丁酸酯（PDBu）和1,2 - 二辛酰甘油（DOG）均能有效抑制血清刺激的DNA合成和细胞群体倍增。当在G1晚期施加时，PDBu对DNA合成的作用最大。PDBu和DOG均不抑制用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）孵育24小时的细胞中的DNA合成，PMA可下调PKC。此外，长时间暴露于PMA会缩短G1期和细胞群体倍增时间。因此，可被佛波酯激活并因长时间暴露于PMA而下调的PKC同工型应参与G1/S抑制。对从G1晚期细胞中提取并通过阴离子交换和羟基磷灰石色谱分离的可溶性蛋白质进行的PKC酶活性测定表明，以PKC -α洗脱的活性占主导，而以PKC -ζ洗脱的活性也可检测到。前者依赖于Ca2+和佛波酯，而后者则不依赖。PKC -ζ似乎以分子量为70 kDa和80 kDa的两个亚类形式表达。在用PMA孵育24小时的细胞中，以PKC -α洗脱的活性完全消失，而以PKC -ζ（70 kDa）洗脱的显著活性仍然存在。另一方面，从颗粒部分以PKC -ε洗脱的是相对较低的、不依赖Ca2+的活性。尽管没有完全消除，但长时间暴露于PMA会使其降低。因此，PKC -α和 -ε可能是G１/S抑制最可能的介质。
