Mitogenic signal of epidermal growth factor in chick embryo hepatocytes: role of PKCalpha

In the present study we investigated the role of two isoforms of protein kinase C in the mitogenic signal of epidermal growth factor (EGF) in primary culture of chick embryo hepatocytes. The down-regulation of PKCalpha by long-term exposure to phorbol myristate acetate (PMA) provoked a reduced mitogenic response to EGF while the down-regulation of PKCepsilon with oligonucleotide antisense had no effect on the stimulation of DNA synthesis, assayed as thymidine incorporation. EGF enhanced H3 diacylglycerol (DAG) production by cells preincubated with H3myristic acid, but did not increase the production of inositol 1-4-5-trisphosphate (IP3). EGF produced an increase in the release of arachidonic acid and prostaglandin E2 (PGE2) in the extracellular medium. The increase was blocked by specific inhibitors (quinacrine and AACOCF3) of phospholipase A2 (PLA2) and was inhibited by down-regulation of PKCalpha, demostrating that this isoform is involved in arachidonic acid production. DAG and arachidonic acid produced an additional effect on thymidine incorporation. The treatment with PLA2 inhibitors, which block the increase in arachidonic acid, decreased the effect of EGF on DNA synthesis. These results suggest that in chick embryo hepatocytes PKCalpha is the main isoform involved in EGF-induced DNA synthesis. Its rapid activation is dependent on DAG production and induces an increased production of arachidonic acid and prostaglandin which are involved in the mitogenic activity.