PKC Preferentially Activates Capacitative Calcium Entry in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: To define the specific roles of protein kinase C (PKC) isozymes in epidermal growth factor (EGF)–induced activation of capacitative calcium entry (CCE) in human corneal epithelial cells (HCEC). Methods: Changes induced by 1 µM PDBu or 20 ng/ml EGF were characterized in localization of six different PKC isoforms using differential centrifugation and Western blotting. Calcium transients in Fura–2 loaded HCEC resulting from exposure to each of these stimulators were monitored with single cell fluorescence imaging. A calcium addback protocol evaluated CCE. Mn²⁺ quenching was used as an index of Ca²⁺ influx. PKC (α, ßI, ßII, Δ, ε and µ) isoform involvement was evaluated based on the relative inhibitory effects of 3 µM rottlerin, 50 µM HBDDE, 2 µM hispidin, and 10 µM V1–2, respectively. Results: Following exposure to PDBu for 30 min, the PKC (α, ßI, Δ and ε) isoforms were differentially translocated. PKCΔ predominately moved from a cytosolic to a plasma membrane enriched fraction. Their rank order of translocation was Δ > ßI > α ∼ε. This order of translocation was in agreement with the magnitudes of inhibition of CCE caused by relatively selective PKC isoform inhibitors. On the other hand, following exposure to EGF, the rank order of PKC isoform translocation to the plasma membrane fraction was: Δ∼ßI > ε >α. The magnitudes of inhibition of EGF–induced CCE caused by the aforementioned inhibitors were similar to the extents of PKC isoform translocation. However, no changes in localization were observed for either ßII or µ. The magnitudes of suppression of EGF and PDBu–induced increases in Mn²⁺ quench by PKC isoform inhibitors mirrored differential declines in CCE caused by these agents. Conclusions: PKCΔ plays a major role in eliciting CCE increases in response to direct stimulation of PKC. On the other hand, EGF activates both the Δ and ßI isoforms to cause this effect. This disparity could be a consequence of differences in the cell signaling pathways activated by PDBu and EGF.

Keywords: calcium • cornea: epithelium • signal transduction