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EGF Stimulates Growth by Enhancing Capacitative Calcium Entry in Corneal Epithelial Cells

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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 [Ca2+]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 of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

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Acknowledgements

The authors deeply appreciate the outstanding support of Wei Li who documented TRP localization with confocal microscopy. This work was supported by NIH grant EY04795 (PR).

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Authors and Affiliations

  1. College of Optometry, Biological Sciences, SUNY, 33 West 42nd Street, New York, NY 10036, USA

    H. Yang, Z. Wang, F. Zhang & P. S. Reinach

  2. School of Optometry, Indiana University, Bloomington, IN 47405, USA

    X. Sun

  3. Departments of Cell Biology, Neurology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA

    G. Ning

  4. Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA

    J. Kong

  5. UCLA School Medicine, Harbor Campus, Molecular Medicine, Torrance, CA 90502, USA

    L. Lu

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  1. H. Yang
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Correspondence to P. S. Reinach.

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Yang, H., Sun, X., Wang, Z. et al. EGF Stimulates Growth by Enhancing Capacitative Calcium Entry in Corneal Epithelial Cells . J. Membrane Biol. 194, 47–58 (2003). https://doi.org/10.1007/s00232-003-2025-9

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