EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.
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 microM 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 microM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 microM UO126 (MEK-1/2 inhibitor) or 10 microM 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 Erkl/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.
D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers; D004533:Egtazic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011817:Rabbits
D015220:Calcium Channels; D017931:DNA Primers; D004533:Egtazic Acid; D004815:Epidermal Growth Factor