title: EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.
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 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.
mesh_terms: 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
chemical_list: D015220:Calcium Channels; D017931:DNA Primers; D004533:Egtazic Acid; D004815:Epidermal Growth Factor