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G.D. Sharma, H.E. Bazan; Differential Translocation Patterns of Green Fluorescent Protein-fused PKC (GFP-PKC) upon Stimulation with Growth Factors in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3812.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Studies from our laboratory had shown that protein kinase C (PKC)α is activated after corneal injury and that inhibition of its expression delays epithelial wound healing. PKC isoforms play crucial roles in intracellular signaling and display differential patterns of subcellular distribution/localization upon various physiologic stimuli. We have transfected green fluorescent protein fused with PKCα (GFP-PKCα) into human corneal epithelial (HCE) cells to examine its redistribution in real time when stimulated with growth factors. Methods: HCE cells were transfected with GFP-PKCa using FuGENE6. After transfection cells were starved overnight in medium devoid of growth supplements Dishes were secured on a temperature-controlled stage at 37 °C and stimulated with hepatocyte, keratinocyte, or epidermal growth factor (HGF, KGF (20 ng/ml), or EGF (10 ng/ml). In some experiments the lipoxygenase metabolite 15 (S) HETE (2 µM), a specific PKCa inhibitor Go6976 (20 nM), or the EGF receptor antagonist PD153035 (10 mM) was used. Images were recorded by software-controlled shutter (Meta Vue) and a Sony digital camera attached to a fluorescence microscope. Results: Cells transfected with GFP-PKCα showed initial expression in the cytoplasm. Treatment with HGF produced GFP-PKCα movement to plasma membrane. The translocation was noticeable 15 minutes after stimulation and more evident at 60 minutes. When cells were pretreated with Go6926 and co-stimulated with HGF, the translocation was inhibited. EGF stimulation was more potent than that of HGF, but GFP-PKCα accumulated in plasma membrane at similar times. Incubations with 15 (S) HETE in the presence of EGF induced a more potent and rapid translocation of GFP-PKCα evident at 5 minutes after stimulation. Cells pre-treated with PD153035 and then stimulated with EGF and monitored for 60 minutes did not show translocation. Unlike the other two growth factors, KGF did not induce GFP-PKCα translocation monitored up to 60 minutes. However, as a positive control, treatment with the phorbol ester TPA (200 nM) after KGF stimulation induced accumulation of GFP fluorescence in plasma membrane within 10 minutes Conclusions: HGF and EGF stimulation produced a relatively slow translocation of PKCα to plasma membrane. 15 (S) HETE accelerated and potentiated the EGF response. KGF did not stimulate PKCα translocation. The differential responses of PKCα movement to the three growth factors suggest that the activation of this kinase isoform could lead to selective responses that promote corneal wound healing.
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