TRPC4 Knockdown Suppresses EGF–Induced Capacitative Calcium Entry and Growth in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: The mitogenic response to epidermal growth factor (EGF) in corneal epithelial cells is dependent on capacitative calcium entry (CCE). The transient potential protein isoform (TRPC4) in corneal epithelial cells is a component of the plasma membrane store operated channel (SOC) activated by intracellular store (ICS) depletion. To determine whether this response is dependent on TRPC4 expression, we knocked it down in human corneal epithelial cells (HCEC). Methods: siRNAs for TRPC4 knockdown were transfected into HCEC. RT–PCR and immunoblot analysis assessed knockdown efficiency of its gene and protein expression. Whole cell patch clamp technique was used to monitor TRPC4 activation following CCE stimulation by ICS depletion. [³H] thymidine incorporation evaluated EGF–induced mitogenesis. [Ca²⁺]_i was measured with single–cell fluorescence imaging. Results: TRPC4 knockdown decreased mRNA and protein expression by 88% and 75%, respectively. ICS depletion induced increases in currents through SOC were eliminated. EGF–induced increases in [Ca²⁺]_i and cell proliferation were decreased by 80% and 54%, respectively. Conclusions: siRNA knockdown of TRPC4 expression inhibited EGF–linked activation of SOC, CCE and cell growth. Therefore, the optimal mitogenic response to EGF depends on store depletion–induced stimulation of SOC.

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