The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.