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

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

Research output: Contribution to journalArticle

36 Citations (Scopus)

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
StatePublished - Jul 1 2003

Fingerprint

Epidermal Growth Factor
Epithelial Cells
Calcium
Growth
Cell Membrane
Rabbits
Protein Isoforms
Calcium-Transporting ATPases
Optical Imaging
Mitogen-Activated Protein Kinase Kinases
Colforsin
Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases
Adenylyl Cyclases
Endoplasmic Reticulum
Thymidine
Electron Microscopy
Fluorescence
Immunohistochemistry
Gene Expression

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Yang, H. ; Sun, X. ; Wang, Z. ; Ning, Gang ; Zhang, F. ; Kong, J. ; Lu, L. ; Reinach, P. S. / EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells. In: Journal of Membrane Biology. 2003 ; Vol. 194, No. 1. pp. 47-58.
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EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells. / Yang, H.; Sun, X.; Wang, Z.; Ning, Gang; Zhang, F.; Kong, J.; Lu, L.; Reinach, P. S.

In: Journal of Membrane Biology, Vol. 194, No. 1, 01.07.2003, p. 47-58.

Research output: Contribution to journalArticle

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AU - Yang, H.

AU - Sun, X.

AU - Wang, Z.

AU - Ning, Gang

AU - Zhang, F.

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AU - Reinach, P. S.

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