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. 2009 Jan 1;46(1):62-9.
doi: 10.1016/j.freeradbiomed.2008.09.021. Epub 2008 Oct 2.

"V体育平台登录" Sustained oxidative stress inhibits NF-kappaB activation partially via inactivating the proteasome

Mingxing Wu  1 , Qingning Bian, Yizhi Liu, Alexandre F Fernandes, Allen Taylor, Paulo Pereira, Fu Shang
Affiliations

Sustained oxidative stress inhibits NF-kappaB activation partially via inactivating the proteasome

VSports手机版 - Mingxing Wu et al. Free Radic Biol Med. .

Abstract

NF-kappaB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation, and stress responses. Many studies indicate that NF-kappaB is a stress-sensitive transcription factor and its activation is regulated by reactive oxygen species VSports手机版. In previous studies, we and others demonstrated that this transcription factor can be activated by transient oxidative stress. However, the effects of sustained oxidative stress on NF-kappaB activation are not clear. The objective of this study was to determine the effects of sustained oxidative stress on NF-kappaB activation and to elucidate the signaling events affected by sustained oxidative stress. Human lens epithelial cells (HLEC) that were subjected to 4 h of continuous influx of hydrogen peroxide were used to investigate the effects of sustained oxidative stress on NF-kappaB activation. The data showed that, unlike transient oxidative stress, sustained exposure of HLEC to physiologically relevant levels of H(2)O(2) (50-100 microM for 4 h) did not induce the degradation of I-kappaB and activation of NF-kappaB, but attenuated TNFalpha-induced degradation of I-kappaB and activation of NF-kappaB. Sustained exposure of HLEC to these levels of H(2)O(2) also inactivated proteasome activity by 50-80%. Consistent with the role of the proteasome in degradation of I-kappaB and activation of NF-kappaB, treatment of HLEC with proteasome inhibitors also attenuated TNFalpha-induced I-kappaB degradation and NF-kappaB activation. The data also indicate that activation of NF-kappaB is essential for the cells to recover from oxidative stress. Inhibiting NF-kappaB activation during recovery from transient oxidative stress significantly reduced the cell viability. Together, these data indicate that sustained oxidative stress may inactivate the proteasome and subsequently inhibit NF-kappaB activation by impeding the degradation of I-kappaB. The oxidative inactivation of the proteasome and subsequent impairment of NF-kappaB activation may contribute to the death of lens epithelial cells, a common feature associated with cataract. .

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Fig. 1
Fig. 1. Continuous influx of H2O2 down regulates TNFα-induced NF- κB activation
Confluent HLEC were cultured in the presence of 0, 20 and 40 mU/ml glucose oxidase for 4 hours. H2O2 levels in the medium were determined every hour during the incubation (panel A). This resulted in maximal concentrations of H2O2 in the medium of 0, 50 and 100 μM, respectively. The cells were then treated with or without 10 ng/ml TNFα in an oxidant-free medium for 30 min. The levels of NF-κB (p65) in the nucleus and cytosol were determined by Western blotting (panel B). The levels of I-κBα and β-actin in the cytosol were also determined by Western blotting (panel B). The data are representatives of three independent experiments.
Fig. 2
Fig. 2. Sustained exposure to H2O2 attenuates TNFα-induced NF- κB activity
Confluent lens epithelial cells were cultured in the presence and absence of 40 mU/ml glucose oxidase for 4 hours. The cells were then treated with 10ng/ml TNFα for 0, 15 and 30 min. The DNA-binding activity of NF-κB in the nuclear extracts was determined by electrophoretic mobility shift assays (upper panel). The levels of I-κBα and β-actin in the cytosol were determined by Western blotting (middle and lower panels, respectively). The data are representatives of three independent experiments.
Fig. 3
Fig. 3. Sustained exposure to H2O2 inactivates the proteasome
Confluent lens epithelial cells were cultured in the presence and absence of 40 mU/ml glucose oxidase for 4 hours. The cells were collected and lysed in a hypotonic buffer with vortexing. After centrifugation the resultant supernatants were used to determine activities of proteasome (panel A) and lactate dehydrogenase (panel B). Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase (PGPH) peptidase activity of the proteasome were determined using fluorogenic peptides as substrates. Enzymatic kinetics were measured with a temperature-controlled microplate fluorometric reader. Excitation/emission wavelengths were 380/440 nm. *P<0.01 in comparison between control and chronically oxidized cells. The data are summarized from two independent experiments, each was done in triplicates.
Fig. 4
Fig. 4. Proteasome inhibition down-regulates TNFα-induced NF- κ B activity
Confluent lens epithelial cells were first cultured in the presence and absence of 10 μM MG132 for 30 min. This concentration of MG132 inhibited the three peptidase activities of the proteasome by 60-85% (panel A). The cells were then treated with10 ng/ml TNFα for 0, 15 and 30 min. The DNA-binding activity of NF-κB in the nuclear extracts was determined by electrophoretic mobility shift assays (panel B, upper panel). Levels of I-κBα and β-actin in the cytosol were determined by Western blotting (panel B, middle and lower panels). The data are representatives of three independent experiments.
Fig. 5
Fig. 5. Sustained exposure to H2O2 also impairs the TNFα-induced phosphorylation of I-κBα
Confluent lens epithelial cells were cultured in the presence or absence of 40 mU/ml glucose oxidase for 4 hours. The cells were then incubated with10 ng/ml TNFα for 0, 5 10 and 30 min. Levels of phosphorylated I-κBα and phosphotylated Akt in the cells were determined by Western blotting analysis using a monoclonal antibody against phosphorylated I-κBα (Ser32/36) or rabbit polyclonal antibodies against phosphorylated Akt (Thr473). The levels of total I-κBα, Akt and β-actin were determined and used as the loading controls. The data are representatives of three independent experiments.
Fig. 6
Fig. 6. Inhibition of NF-κB activation during recovery from transient oxidative stress potentiates cytotoxicity of oxidative stress
Subconfluent HLEC were treated with indicated concentrations H2O2 for 1 h and then allowed to recover in oxidant-free medium for 16 hour in the absence or presence of 5 μM BAY 11-7082, or 5 μM BMS-345541, two classic NF-κB inhibitors. The cell viability was determined by MTS assay. Panel A, MTS activity immediately after exposure to H2O2. Panel B, the effects of MG132, BAY 11-7082, and BMS-345541 on NF-κB activation. Panel C, MTS activities after 16 hour recovery in the presence or absence of 5 μM BAY 11-7082. Panel D, MTS activities after 16 hour recovery in the presence or absence of 5 μM BMS 345541. Data in panels A, C and D are summary of two independent experiments, each done in triplicates. * indicates p <0.05 and ** indicates p<0.01 when compared with cells treated with same levels of H2O2. The data in panel B are representatives of two independent experiments.
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