EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells

H. Yang, X. Sun, Z. Wang, G. Ning, F. Zhang, J. Kong, L. Lu, P. S. Reinach

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Abstract

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca2+]i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca2+]i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 μM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca2+ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn2+ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 μm 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 μM UO126 (MEK-1/2 inhibitor) or 10 μM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca 2+]i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP3R isoforms 1-3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [3H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erk1/2 activity, whereas PKA stimulation suppresses these effects ofEGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

Original languageEnglish (US)
Pages (from-to)47-58
Number of pages12
JournalJournal of Membrane Biology
Volume194
Issue number1
DOIs
Publication statusPublished - Jul 1 2003

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Physiology
  • Cell Biology

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