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M. Wu, Q. Bian, A. F. Fernandes, A. Taylor, P. Pereira, F. Shang; Chronic Oxidative Stress Inhibits NF-B Activation in Human Lens Epithelial Cells Partially via Inhibiting the Ubiquitin-Proteasome Pathway. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2277.
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© ARVO (1962-2015); The Authors (2016-present)
NF-ΚB is important transcription factor for cell survival, proliferation and transformation. Previous work indicates that transient oxidative stress activates NF-ΚB in lens epithelial cells. The objective of this study is to determine the effect of chronic oxidative stress on signaling events of TNFα-induced NF-ΚB activation and the underlying mechanism.
HLEC (SRA 01/04) were exposed to a continuous flux of hydrogen peroxide produced by glucose and glucose oxidase for 4 hours. TNFα was then added to trigger the activation of NF-ΚB. DNA binding activity of NF-ΚB was determined using electrophoretic mobility shift assay (EMSA). Phosphorylation and degradation of the inhibitor of NF-ΚB (I-ΚB) were determined by Western blotting. Proteasome activity was determined using fluorogenic peptides as substrates.
Unlike transient oxidative stress, chronic exposure of HLEC to physiologically relevant levels of H2O2 (50 µM for 4 hours) did not induce the degradation of I-ΚB and activation of NF-ΚB. However, chronic exposure to H2O2 attenuated TNFα-induced degradation of I-ΚB and activation of NF-ΚB. Chronic exposure of HLEC to these levels of H2O2 also inhibited proteasome activity by 40-50%. Consistent with the role of the ubiquitin-proteasome pathway (UPP) in degradation of I-ΚB and activation of NF-ΚB, treatment of HLEC with proteasome inhibitors also attenuated TNFα-induced degradation of I-ΚB and activation of NF-ΚB.
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