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 hydroxytapatite chromatography showed that the activity eluting with PKC-α predominated, whereas that eluting with PKC-ζ was detectable. The former was dependent on Ca²⁺ and phorbol ester but the latter was not. PKC-ζ 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-α was completely abolished, whereas the significant activity eluting with PKC-ζ (70 kDa) remained. On the other hand, a relatively low, Ca²⁺-independent activity etuted with PKC-ε from the particulate fraction. This was reduced by long exposure to PMA, although not completely. Therefore, PKC-α and -ε may be the most probable mediators of the G₁/S inhibition.