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L. Wang, I.M. Wormstone, J.R. Reddan, G. Duncan; EGF Receptor Signaling in Human Lens Cells: Dependence on a Functional Calcium Store . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4503.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies have shown that a functional calcium store is required for the growth of lens cells. This study investigated the role of the calcium store on the signaling pathway of a human lens cell growth promotor, epidermal growth factor (EGF). Methods: FHL 124 human lens cells were routinely cultured and seeded on coverslips or tissue culture dishes. EGF receptor was identified using immunocytochemistry and visualized using a fluorescent microscope with a cooled CCD camera. Activation of the MAP kinase signaling protein ERK was detected using western blot techniques. Intracellular calcium signaling was disabled using the endoplasmic reticulum CaATPase inhibitor thapsigargin. Results: Cells maintained in serum-free medium possessed EGF receptors that were distributed diffusely across the plasma membrane. Addition of 10ng/ml EGF induced the internalization of receptors visualized by appearance of bright vesicles within the cells. This occurred within 30 minutes of exposure. Furthermore, 10ng/ml EGF significantly stimulated the expression of phosphorylated ERK again within 30 minutes. Maintenance in 100nM thapsigargin for up to 48 hours did not appear to prevent EGFR internalization or ERK phosphorylation following a single exposure to 10ng/ml EGF. However, when cells maintained in thapsigargin were exposed to EGF for 30 minute periods at both 24 and 48 hours, the second response was reduced relative to serum-free maintained controls. Conclusion: An intact endoplasmic reticulum calcium store is not apparently required for initial receptor activation, ERK phosphorylation, or receptor internalisation. However, inhibition of calcium signaling by thapsigargin significantly reduces the ability of lens cells to respond to multiple EGF exposures.
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