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H Yang, XC Sun, CB Zhai, M Cui, JA Bonanno, Z Wang, PS Reinach; EGF Stimulates Growth Through Calcium-induced Calcium Release Activation in Rabbit Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1652.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We validated in SV40-immortalized rabbit corneal epithelial cells (tRCEC) that EGF induced calcium transients are accounted for by calcium-induced calcium release (CICR) and that they are needed for EGF receptor mediated control of proliferation. Methods: After fura2-AM loading, [Ca2+]i fluorescence ratio cell imaging was performed. Reverse transcription- polymerase chain reaction (RT-PCR) was used to probe for the expression of transient receptor potential (TRP) isoforms (TRP1-7) and Inositol 1,4,5-trisphosphate receptor (IP 3 R) subtypes (IP3R1-3). Subcloning and sequencing were performed to confirm their identity. [3H]-thymidine incorporation was done to measure proliferation following 24 h serum starvation. Results: To evaluate whether EGF-induced calcium transients depend on store operated channel (SOC) stimulation, Mn2+ quenching of fura2 fluorescence was done. Under control conditions the slope of the decline in fluorescence was -0.026 ± 0.2/ 5 sec. EGF (5 ng/ml: optimal dose for growth stimulation) increased it to -1.23 ± 0.1/5 sec. RT-PCR analysis gave positive bands at the predicted size for TRP1 (397 bp); TRP3 (624 bp); TRP4 (602 bp); TRP6 (425 bp); TRP7 (326 bp), and IP3R1 (1070 bp); IP3R2 (1335 bp); IP3R3 (211 bp). However, the RT-PCR analysis for TRP2 and TRP5 was always negative. Sequencing further confirmed the specificities of all detected PCR products. The blockade of plasma membrane SOC activity with 100 µM 2-APB significantly suppressed the proliferative effect of EGF. Conclusion: EGF induced increases in Mn2+ fluorescence quenching kinetics and identification of TRP and IP3 isoform gene expression affirm that this cytokine induces calcium transients through CICR. As inhibition of SOC activity diminishes the proliferative response to EGF, CICR activity is essential for this mitogenic effect.
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