Differential roles of PKC isoforms in enhancing Store Operated Channel activity in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: Epidermal growth factor (EGF), in corneal epithelial cells, stimulates protein kinase C (PKC) activity and capacitative calcium entry (CCE). We determined in these cells the differential roles of PKC isoforms in mediating those effects of EGF. Methods: Single cell fluorescence imaging was performed on fura2–AM loaded human corneal epithelial cells (HCEC) on the stage of an inverted microscope. CCE was activated by first depleting intracellular calcium in Ca²⁺–free medium containing 0.5 mM EGTA and inhibiting endoplasmic reticulum calcium pump activity with 5 µM cyclopiazonic acid (CPA). Plasma membrane calcium influx through pathways that include store operated channels (SOC) was evaluated based on increases in [Ca²⁺]_i resulting from 1 mM calcium addback to the bathing medium. Relatively selective PKC isoform inhibitors were used to suppress the stimulation of SOC in the presence and absence of 1 µM PDBu. Immunocytochemistry and confocal microscopy were performed to detect the intracellular translocation of different PKC isoforms during the stimulation of CCE. Results: Ca²⁺ addback caused a transient increase in F₃₄₀/F₃₈₀ nm of 0.74 units within 30 s. Either 1 µM PDBu or 20 ng/ml EGF each increased this response 1.3–fold whereas 1 µM 4α–phorbol didecanoate had no effect. Preincubation with either 1 µM PMA for 24h or pan–PKC inhibitor, 1 µM calphostin C for 30 min obviated both PDBu and EGF induced stimulation of addback. Similarly, the PKC α inhibitor, 50 µM HBDDE and the PKCα, ß inhibitors, 0.05 µM Go6976 and 0.2 µM GF109203X abolished the effect of PDBu on addback. Furthermore, Go6976 and GF 109203X fully suppressed the EGF stimulated calcium addback response. However, the PKC ß inhibitor, 2 µM hispidin, and the PKCε translocation inhibitory peptide 10 µM V1–2 only partially suppressed this effect. With a PKC δ inhibitor, 3 µM rottlerin, the addback increment declined below the control level and that observed following PKC downregulation. Either 1 µM PMA or activation of CCE with CPA resulted in PKCα and δ relocalization from the cytosol to the perinuclear area and plasma membrane. Conclusions: Specific PKC isoforms mediate EGF–induced stimulation of SOC activity. This is indicated because: 1) PKC downregulation or calphostin C obviated both PDBu and EGF induced increases in addback; 2) Five (i.e. PKC α, ßI, ßII, δ and ε) out of the six PKC isoforms identified in HCEC mediate this effect of PDBu on addback. The largest contributor is PKC α.

Keywords: calcium • signal transduction • cornea: epithelium