Endothelin-1 stimulates DNA synthesis and anchorage-independent growth of Rat-1 fibroblasts through a protein kinase C-dependent mechanism

Stimulation of quiescent cultured fibroblasts with a variety of growth-promoting factors induces release of diacylglycerol (DG) and subsequent activation of protein kinase C (pkC), but the role of pkC in the induction of DNA synthesis and cell proliferation remains unclear. We have investigated the involvement of pkC in the response of Rat-1 fibroblasts to the newly described peptide endothelin-1 (Et-1), an agonist that is secreted by the vascular endothelium and that may play a role in the proliferative response of cells in the vessel wall. Addition of Et-1 to serum-deprived Rat-1 cells promoted DNA synthesis in the absence of additional factors and stimulated anchorage-independent growth in the presence of epidermal growth factor (EGF), indicating that Et-1 has many of the characteristics of a mitogen. The ability of Et-1 to stimulate both DNA synthesis and anchorage-independent growth was markedly reduced by the depletion of cellular pkC activity induced by prolonged exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, the ability of Et-1 to induce both second messenger production and transcription of c-fos and c-jun was largely independent of cellular pkC activity. Production of DG in response to Et-1 persisted for >12 h and may account for the ability of Et-1 to augment the G₁-S phase transition. Although these observations indicate that functional pkC is not an essential component of the proximal pathway leading to rapid changes in gene transcription and second messenger production in response to Et-1 treatment, the data suggest that activation of pkC is an essential component of the downstream events responsible for the stimulation of cell proliferation and anchorage-independent growth in Rat-1 cells exposed to Et-1.