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Y. Wang, L. Lu; Ctcf Activation in Egf-Induced Corneal Epithelial Proliferation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6239.
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The aims of the present study are to investigate the mechanism(s) by which upstream components of the NF-ΚB signaling pathway mediate EGF- and UV stress-induced CTCF activation determining human corneal epithelial (HCE) fate.
IKKα and IKKβ, as well as their kinase-silencing mutants (IKKα-KM /IKKβ-KM) were overexpressed in HCE cells, respectively.HCE cells were synchronized by serum-deprived culture for 48 h prior to experimental treatments. EGF (20 ng/ml) and/or UV irradiation (245 nm at 42 µJ/cm2) were applied to HCE cells. CTCF activities were evaluated by luciferase reporter assay, and the protein levels of CTCF were detected by western blot. NF-ΚB dimers formed by different subunits in EGF- and UV-induced cells were verified by immunoprecipitation experiments.
We have examined the effects of IKKα and IKKβ upstream from the NF-ΚB pathway on EGF-induced cell proliferation and UV stress-induced apoptosis through regulating CTCF activities. Results from CTCF-specific luciferase reporter assays in IKKβ over-expressed cells showed that CTCF activities were enhanced and decreased by EGF stimulation and UV stress, respectively. Inhibition of IKKβ by overexpression of its kinase-silencing mutant significantly abolished EGF- and UV-induced effects on CTCF activities. However, IKKα, the other important regulator of the NF-ΚB pathway, had no effect on EGF- and UV stress-induced changes of CTCF activity in cells that were transfected with wildtype IKKα and kinase-silencing IKKα mutant.
These results indicate that IKKβ, an upstream kinase of the NF-ΚB signaling pathway, is responsible for regulation of CTCF in determining EGF- and UV stress-induced corneal epithelial cell fate.
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