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H. Yang, Z. Wang, P. Reinach; Dependence of the Mitogenic Response to EGF on Capacitative Calcium Entry in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3808.
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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.
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