Evidence that reactive oxygen species do not mediate NF-kappaB activation - PubMed (original) (raw)

Evidence that reactive oxygen species do not mediate NF-kappaB activation

Makio Hayakawa et al. EMBO J. 2003.

Abstract

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kappaB activation. This hypothesis is mainly based on the findings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kappaB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kappaB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IkappaB-ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kappaB signaling, but instead lower the magnitude of its activation.

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Figures

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Fig. 1. Effect of compounds with antioxidant properties on TNF- induced NF-κB activation in Jurkat T cells. (A) Jurkat T cells were pretreated with either 30 mM NAC or 200 µM PDTC for 1 h, and then stimulated with 3 ng/ml TNF for the indicated times, and nuclear and cytoplasmic extracts were prepared. Nuclear extracts were used to detect NF-κB DNA binding by EMSA. Using the same nuclear extracts, EMSA for constitutively DNA-binding protein Oct-1 was carried out as a loading control. The cytoplasmic extracts were analyzed by immunoblotting to detect IκBα levels. (B) Jurkat T cells were pretreated with the indicated concentrations of either EGCG or Trolox for 1 h, and then stimulated or left untreated with 3 ng/ml TNF for 20 min. EMSA and immunoblotting were carried out as described in (A).

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Fig. 2. NAC selectively inhibits TNF-activated signaling pathways. (A) HeLa cells were pretreated with 30 mM NAC for 1 h, followed by stimulation with either 3 ng/ml TNF or 50 ng/ml TPA for the indicated times. Immunoblotting analyses were performed to detect IκBα, phospho-p38 and phospho-p44/42 ERKs, respectively. c-Jun N-terminal kinase activity was measured using GST–c-Jun (1–79). (B) HeLa cells were pretreated with 30 mM NAC for 1 h, followed by stimulation with either 1 ng/ml IL-1 or 3 ng/ml TNF for the indicated times. Immunoblotting analyses were performed to detect IκBα, phospho-JNKs, phospho-p38 and phospho-p44/42 ERKs. N.S., non-specific.

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Fig. 3. NAC attenuates TNF receptor signaling by lowering receptor affinity. (A) L929 cells cultured in 10-cm dishes were pretreated either 30 mM NAC or 200 µM PDTC for 1 h, followed by incubation with 20 ng/ml TNF for the indicated times. Cell extracts were precleared with preimmune serum/protein G–Sepharose and then immunoprecipitated with anti-mouse TNF R1 coupled to protein G–Sepharose, followed by immunoblotting analysis to detect TRAF2. The PVDF membrane was stripped and reprobed to detect RIP. As controls, the anti-mouse TNFR1-coupled beads and the whole-cell extract from non-stimulated L929 cells were directly resolved by SDS–PAGE followed by immunoblotting analyses. (B) L929 cells were incubated with increasing amounts of [125I]TNF in the presence (open circles) or absence (filled circles) of 30 mM NAC at 4°C for 2 h. Specific binding was calculated by subtraction of non-specific binding (in the presence of a 100-fold excess of unlabeled TNF) from total binding. Each point is the mean of duplicate samples (left panel). Scatchard analyses (right panel) yielded the following data: control cells, 2490 sites/cell, _K_d = 0.81 nM; NAC-treated cells, 2170 sites/cell, _K_d = 4.99 nM. B/F, bound/free.

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Fig. 4. Divergent modulation of TNF-, IL-1- and TPA-activated signaling pathways by PDTC. (A) L929 cells were pretreated with the indicated concentrations of PDTC for 1 h, followed by the stimulation with 20 ng/ml TNF for 10 min. The cells were lysed and immunoblotting analyses were performed to detect IκBα, phospho-JNKs, phospho-p38 and phospho-p44/42 ERKs. N.S., non-specific. (B) L929 cells were pretreated 200 µM PDTC for 1 h, followed by the stimulation with 1 ng/ml IL-1 for the indicated times. Immunoblotting analyses were performed as described in (A). (C) HT-29 cells were pretreated with 200 µM PDTC for 1 h, followed by stimulation with 3 ng/ml TNF, 10 ng/ml IL-1 or 100 ng/ml TPA for the indicated times. Immunoblotting analyses were performed as described in (A).

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Fig. 5. PDTC inhibits ubiquitylation of IκBα and β-catenin. (A) L929 and HT-29 cells were pretreated with 200 µM PDTC for 1 h, followed by the stimulation with TNF for the indicated times. Then cytoplasmic and nuclear extracts were prepared as described in Materials and methods. The cytoplasmic extracts were used for IKK assay and immunoblotting analyses to detect phospho-IκBα and IκB. Nuclear extracts were used to measure NF-κB and Oct-1 DNA binding activities by EMSA. (B) L929 cell transiently overexpressing HA-tagged ubiquitin were pretreated with 10 µM MG132 in the presence or absence of 200 µM PDTC for 1 h, followed by stimulation with 3 ng/ml TNF for the indicated times. Cell extracts were immunoprecipitated with anti-IκBα antibodies followed by immunoblotting to detect HA epitope. Then PVDF membrane was stripped and reprobed with anti-IκBα antibody. Ig, immunoglobulin heavy chain. (C) L929 cells transiently overexpressing HA-tagged ubiquitin were pretreated with 10 µM MG132 in the presence or absence of 200 µM PDTC for 2 h. Cell extracts were immunoprecipitated with anti-β-catenin antibodies followed by immunoblotting to detect HA epitope. Then PVDF membrane was stripped and reprobed with anti-β-catenin antibody. (D) HT-29 cell extracts were pretreated as described in (A) and immunoprecipitated with anti-IκBα antibodies followed by immunoblotting to detect endogenous ubiquitin. (E) L929 and HT-29 cell extracts were prepared as described in (B) and immunoprecipitated with anti-β-catenin followed by immunoblotting to detect endogenous ubiquitin.

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Fig. 6. Direct inhibition of Cul1-ROC1-Skp1-FWD1/β-TrCP SCF ubiquitin ligase complex by PDTC. (A) L929 cells overexpressing FLAG-tagged Cul1, HA-tagged FWD1/β-TrCP, Skp1 and ROC1 were harvested in the extraction/washing buffer containing either free EDTA (EDTA) or zinc-saturated EDTA (EDTA-Zn) as described in Materials and methods. Before harvesting the cells, the extraction/washing buffer was supplemented with the indicated concentrations of PDTC. The cell extracts were immunoprecipitated with anti-FLAG antibodies and the resultant immunoprecipitates were analyzed by an in vitro ubiquitylation assay in the presence of the indicated concentrations of PDTC. Separately, the cell extracts were subjected to immunoblotting analyses to detect either FLAG-Cul1 or HA-FWD1/β-TrCP. The bands marked by asterisks are non-specific. (B) L929 cells overexpressing FLAG-tagged Cul1, HA-tagged FWD1/β-TrCP, Skp1 and ROC1 were pretreated with the indicated concentrations of PDTC, followed by stimulation with 20 ng/ml TNF for 15 min. The cells were lysed and the immunoblotting analysis was performed to detect IκBα. (C) Recombinant SCF ubiquitin ligase complex was purified and preincubated with the indicated concentrations of PDTC. Then in vitro ubiquitylation assay was carried out as described in Materials and methods. E3, recombinant SCF complex bound to the beads.

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Fig. 7. TNF-induced NF-κB activation occurs independently of Rac/NADPH oxidase while TPA-induced NF-κB activation depends on Rac. (A) Hygromycin-resistant clonal cell lines derived from HeLa Tet-On cells transfected with pTRE-Myc-N17Rac1 (HeLa Tet-On-N17Rac1 cells) were treated or left untreated with 2 µg/ml Dox for 48 h. The cells were lysed and immunoblotting analyses were performed using either anti-Rac1 antibody or anti-Myc antibody. (B) HeLa Tet-On-N17Rac1 cells were treated with 2 µg/ml Dox for 48 h. The cells were then stimulated with either 3 ng/ml TNF or 3 ng/ml TPA for the indicated times. Nuclear extracts were used to detect NF-κB and Oct-1 DNA binding by EMSA. (C) HeLa Tet-On-N17Rac1 cells were seeded on glass coverslips and treated or left untreated with 2 µg/ml Dox for 48 h. The cells were stimulated with either 3 ng/ml TNF or 3 ng/ml TPA followed by the immunostaining analyses to detect N17Rac1 (Myc) and p65 subunit of NF-κB (p65). (D) HeLa Tet-On-N17Rac1 cells were treated or left untreated with 2 µg/ml Dox for 48 h. The culture medium was replaced with Hank’s balanced salt solution (HBSS) and the cells were stimulated with either 3 ng/ml TNF or 3 ng/ml TPA for the indicated times. ROS production was measured using DCFH-DA as described in Materials and methods. **P <0.01; ***P <0.001.

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Fig. 8. ROS produced through NADPH oxidase do not mediate TPA-induced NF-κB activation. (A) Hygromycin-resistant clonal cells derived from HeLa Tet-On cells transfected with pTRE-Myc-V12Rac1 (HeLa Tet-On-V12Rac1) were treated or left untreated with 2 µg/ml Dox for 48 h. Immunoblotting analyses were performed as described in Figure 6A. (B) HeLa Tet-On-V12Rac1 cells were treated or left untreated with 2 µg/ml Dox for 48 h. The cells were further cultured in the presence or absence of 20 µM DPI for 3 h. The culture medium was replaced with HBSS and the cells were stimulated or left untreated with 3 ng/ml TPA for 30 min in the presence or absence of DPI. ROS production was measured using DCFH-DA as described in Materials and methods. *P <0.05; **P <0.01. (C) HeLa Tet-On-V12Rac1 cells were treated or left untreated with 2 µg/ml Dox for 48 h. The cells were further cultured in the presence or absence of 20 µM DPI for 3 h. The cells were then stimulated with 3 ng/ml TPA for the indicated times. Nuclear extracts were subjected to EMSA to detect NF-κB and Oct-1 DNA binding activities.

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