Chronic Oxidative Stress Inhibits NF-B Activation in Human Lens Epithelial Cells Partially via Inhibiting the Ubiquitin-Proteasome Pathway

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Keywords: oxidation/oxidative or free radical damage • signal transduction • proteolysis