EGF Stimulates Growth by Enhancing Capacitative Calcium Entry in Corneal Epithelial Cells |
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Authors: | H.?Yang,X.?Sun,Z.?Wang,G.?Ning,F.?Zhang,J.?Kong,L.?Lu,P. S.?Reinach author-information" > author-information__contact u-icon-before" > mailto:preinach@sunyopt.edu" title=" preinach@sunyopt.edu" itemprop=" email" data-track=" click" data-track-action=" Email author" data-track-label=" " >Email author |
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Affiliation: | (1) College of Optometry, Biological Sciences, SUNY, 33 West 42nd Street, New York, NY 10036, USA;(2) School of Optometry, Indiana University, Bloomington, IN 47405, USA;(3) Departments of Cell Biology, Neurology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA;(4) Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA;(5) UCLA School Medicine, Harbor Campus, Molecular Medicine, Torrance, CA 90502, USA |
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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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Keywords: | Cell proliferation Epidermal Growth Factor (EGF) Store Operated Channels (SOC) Calcium signaling Cornea Transient Receptor Potential (TRP) |
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