Differential Roles of PKC Isozymes in Mediating EGF Receptor Control of Proliferation in Human Corneal Epithelial Cells.

Abstract

Abstract: : Purpose: Activation by epidermal growth factor (EGF) of extracellular signal–regulated protein kinase (ERK1/2) and specific protein kinase C isoforms is essential for corneal epithelial mitogenesis. In the present study, we investigated, in SV40–immortalized human corneal epithelial cells (HCEC) stimulated by EGF, the differential roles of protein kinase C (PKC) isozymes in activation of the ERK pathway, Na–K–2Cl cotransporter (NKCC1) and cell proliferation. Methods: After 24 h incubation without serum, HCEC proliferation was stimulated with 5 ng/ml EGF. EGF–induced PKCs activation was inhibited by bisindolylmaleimide 1 (competitive inhibitor for the ATP–binding site) or calphostin C (competes for binding at the DAG site). Intracellular Ca²⁺ was chelated with BAPTA–AM. Levels of ERK1/2 and NKCC1 phosphorylation were determined by Western blotting/ECL assay 15 min after EGF exposure. Cell proliferation was determined based on measurements of ³H–thymidine incorporation after 20 h. Results: PKCs inhibition by bisindolylmaleimide 1 (0.1–1.0 µM} in serum starved HCEC produced dose–dependent increases of up to 6–fold, 80% and 25% in NKCC1, ERK1/2 phosphorylation and cell proliferation, respectively. However, there were no significant effects on NKCC1, ERK1/2 activation and the mitogenic response to EGF. On the other hand, calphostin C (1 µM) significantly decreased by more than 30% both ERK1/2 activity and cell proliferation, which completely inhibited the cell response to EGF. In addition, EGF–induced ERK1/2 activation was not very dependent on intracellular Ca²⁺concentration, IC₅₀ = 200 µM BAPTA–AM. Conclusions: In serum–starved HCEC, some PKC isozyme(s) are involved in the control of cell cycle arrest and EGF–induced ERK1/2 stimulation. Its mitogenic effect is independent on the activation of novel Ca²⁺ independent and DAG stimulated PKC isozymes (i.e. δ, ε, &#952, η) and is accompanied by NKCC1 activation, an important component of cell volume regulation. We are currently identifying which of these PKC isozymes are involved in eliciting such effects.

Keywords: cornea: epithelium • signal transduction • growth factors/growth factor receptors