Protein kinase C-mediated inhibition of vascular smooth muscle cell proliferation: the isoforms that may mediate G1/S inhibition.
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.