EGF–Induced Mitogensis in Human Corneal Epithelial Cells (HCEC) Is Dependent on Stimulation by PKC of Store Operated Channel (SOC) Activity

Abstract

Purpose: : TRPC4 protein expression is a component of store–operated channel (SOC) activity mediating epidermal growth factor (EGF)–induced Ca²⁺ transients in SV40–immortalized HCEC (J. Biol. Chem. 2005, 280, 32230–37). As the mitogenic response to EGF is dependent on protein kinase C (PKC) stimulation by this growth factor, we determined here the role of PKC activation in mediating EGF–induced stimulation of SOC activity.

Methods: : TRPC4 mRNA expression was detected by RT–PCR and protein expression was confirmed by western blotting. Ca²⁺ transients were measured by single cell fluorescence imaging. The whole–cell patch–clamp technique was used to monitor EGF–mediated stimulation of SOC activity. PKC activity was inhibited with calphostin C.

Results: : Extracellular application of EGF (20 ng/ml) elicited increases in non–selective cation channel currents in HCEC from 36 ± 6 pA/pF to 98 ± 15 pA/pF (± SEM; n = 3; p < 0.05). In addition, after 15 min, [Ca²⁺]_i increased from 102 ± 1 % nM of its control (base level was set to 100%) to a peak level of 107 ± 2 % (n = 4; p < 0.05). In contrast, pre–incubation with 1 µM calphostin C abolished EGF–induced increases in such currents (47 ± 17 pA/pF vs. 51 ± 23 pA/pF; n = 3; p = 0.89). Interestingly, calphostin C also decreased the [Ca²⁺]_i below its base level to 97 ± 2 % of control (n = 3; p < 0.01).

Conclusions: : EGF elicits [Ca²⁺]_i transients and increases in SOC activity. These effects are dependent on EGF–induced stimulation of PKC activity since calphostin C attenuated such rises. These results and those from previous studies show that EGF–induced increases in proliferation and cell migration are dependent on PKC stimulation of SOC activity by this mitogen.

Keywords: cornea: epithelium • ion channels • growth factors/growth factor receptors