Dependence of the Mitogenic Response to EGF on Capacitative Calcium Entry in Corneal Epithelial Cells

Abstract

Abstract: : Purpose: To determine whether capacitative calcium entry (CCE) is involved in eliciting the mitogenic response to epidermal growth factor, EGF in SV40-immortalized rabbit corneal epithelial cells (RCEC). Methods: [³H] thymidine incorporation characterized cell proliferation. [Ca²⁺]_i was measured by single cell fluorescenceimaging. Immunogold electron and confocal microscopy detected TRP localization. Results: EGF (5 ng/ml) maximally increased [Ca²⁺]_i 4-fold. Depletion of intracellular store (ICS) Ca²⁺ with 10 µM cyclopiazonic acid (CPA), followed by Ca²⁺ addback stimulated store operated channel (SOC) activity. Such activity was blocked with 100 µM 2-aminoethyldiphenylborate (2-APB). On the other hand, exposure to either 50 µM UO126 or 10 µM forskolin, eliminated the subsequent effect of EGF in raising [Ca²⁺]_i. Confocal and immunogold electron microscopy detected plasma membrane localization of TRP4. Inhibition of CCE with 2-APB and/or CPA eliminated the 2.5-fold increase in intracellular [³H]-thymidine incorporation induced by EGF. Conclusions: EGF induced calcium mobilization and increases in cell growth require both Erk1/2 stimulation and SOC activation. SOC involvement is evident because:1) 2-APB inhibited ICS calcium refilling; 2) TRP4 plasma membrane localization was observed, which is a hallmark of SOC activity.

Keywords: cell-cell communication • cell membrane/membrane specializations • calcium