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L. Lu, T. Li; EGF-Induced Corneal Epithelial Proliferation Mediated by CTCF Through Activation of NF-B. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2952.
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Our previous study indicates that CTCF, CCCTC nuclear factor, plays a central role in regulating Pax6 in EGF-induced proliferation. In the present study, we investigate further the role of CTCF in downstream from the EGF-induced NF-ΚB signal pathway to promote human corneal epithelial (HCE) cell proliferation.
HCE cells were cultured in DMEM/F12 medium with 10% FBS and 5 µg/ml insulin. EGF-induced proliferation was evaluated by MTT assay. CTCF expression was measured by western blots. Activation CTCF was determined by measuring CTCF promoter activity. NF-ΚB activation was determined by electrophoresis of gel migration shift assay (EMSA). The effects of NF-ΚB subtypes (p50 and p65) on CTCF transcription were analyzed by EMSA and their nuclear translocations.
We found that EGF-induce proliferation was mediated by CTCF in HCE cells by enhancing CTCF promoter activity, suggesting that EGF regulates CTCF through a transcriptional mechanism. Blockade of EGF-activated NF-ΚB with PDTC effectively suppressed EGF-induced CTCF expression and proliferation. EGF-induced activation of CTCF is dependent on the formation of NF-ΚB dimers that consist with p65 and p50 subtypes. The results were further verified by p50 and p65 nuclear translocation in response to EGF stimulation. Knockdown of p50 and p65 with specific siRNAs resulted in inhibition of EGF-induced CTCF expression and retardation of proliferation.
Our results reveal for the first time that CTCF plays a significant role in downstream of the EGF-stimulated NF-ΚB signal pathway to regulate proliferation in corneal epithelial cells.
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