Oxidants positively or negatively regulate nuclear factor kappaB in a context-dependent manner

Abstract

Redox-based mechanisms play critical roles in the regulation of multiple cellular functions. NF-kappaB, a master regulator of inflammation, is an inducible transcription factor generally considered to be redox-sensitive, but the modes of interactions between oxidant stress and NF-kappaB are incompletely defined. Here, we show that oxidants can either amplify or suppress NF-kappaB activation in vitro by interfering both with positive and negative signals in the NF-kappaB pathway. NF-kappaB activation was evaluated in lung A549 epithelial cells stimulated with tumor necrosis factor alpha (TNFalpha), either alone or in combination with various oxidant species, including hydrogen peroxide or peroxynitrite. Exposure to oxidants after TNFalpha stimulation produced a robust and long lasting hyperactivation of NF-kappaB by preventing resynthesis of the NF-kappaB inhibitor IkappaB, thereby abrogating the major negative feedback loop of NF-kappaB. This effect was related to continuous activation of inhibitor of kappaB kinase (IKK), due to persistent IKK phosphorylation consecutive to oxidant-mediated inactivation of protein phosphatase 2A. In contrast, exposure to oxidants before TNFalpha stimulation impaired IKK phosphorylation and activation, leading to complete prevention of NF-kappaB activation. Comparable effects were obtained when interleukin-1beta was used instead of TNFalpha as the NF-kappaB activator. This study demonstrates that the influence of oxidants on NF-kappaB is entirely context-dependent, and that the final outcome (activation versus inhibition) depends on a balanced inhibition of protein phosphatase 2A and IKK by oxidant species. Our findings provide a new conceptual framework to understand the role of oxidant stress during inflammatory processes.

FIGURE 1.

Oxidants and TNFα activate NF-κB with different time courses. A549 cells were stimulated with 10 ng/ml TNFα or 500 μm PN. A and D, cytoplasmic or nuclear p65 as well as tubulin were detected by Western blotting. B and E, NF-κB-DNA binding activity was determined by EMSA. C and F, IκBα levels were determined by Western blotting. G, IKK activity was determined by the IKK assay (KA). H and I, total and phosphorylated IKKα/β were determined by Western blotting.

FIGURE 2.

Postexposure to oxidants amplifies TNFα-dependent NF-κB activation. A549 cells were unstimulated (control, c) or stimulated with decomposed PN for 1 h (DP), with 500 μm PN for 1 h, with 10 ng/ml TNFα for 30 min followed by 0–500 μm PN for 1 h (PN post-TNF), or with 10 ng/ml TNFα for 30 min followed by PBS buffer for 1 h (PBS post-TNF). A, NF-κB-DNA binding activity was determined by EMSA and was quantified by densitometry (*, p < 0.05 versus c. †, p < 0.05 versus TNFα + PN 0). B and C, nuclear p65 and nuclear phosphorylated p65 were determined by Western blotting. D, A549 cells were transiently co-transfected with a plasmid bearing NF-κB-firefly luciferase reporter gene and with a plasmid bearing Renilla luciferase. Transfected cells were unstimulated (c), stimulated with 10 ng/ml TNFα for 30 min followed by PBS (PBS post-TNFα) or by 500 μm PN (PN post-TNF) for 1 h and then replaced in medium for 3 additional h. Luciferase activity was measured in cell lysates (*, p < 0.05). E, IL-8 (enzyme-linked immunosorbent assay) in the medium of unstimulated cells (control c), or after stimulation with 10 ng/ml TNFα (30 min) followed by PBS for 4 h (PBS post-TNF) or with 10 ng/ml TNFα (30 min) followed by 500 μm H₂O₂ for 4 h (H2O2 post-TNF), or with 500 μm H₂O₂ alone for 4 h (*, p < 0.05) is shown. F, L929 cells were unstimulated (control, c) or were stimulated with 10 ng/ml TNFα for 1 h, with 25 mm N-acetylcysteine for 1 h followed by 10 ng/ml TNFα for 1 h (NAC+TNF) or with 25 mm N-acetylcysteine alone for 1 h (NAC). NF-κB-DNA binding activity was determined by EMSA. Error bars indicate S.E.

FIGURE 3.

Postexposure to oxidants prevents IKK dephosphorylation, leading to prolonged activation of IKK and sustained IkBα degradation. A549 cells were stimulated with 10 ng/ml TNFα for 10 min followed by 500 μm peroxynitrite (PN post-TNFα) or PBS-glucose buffer (PBS post-TNFα) for the indicated times. A, phospho-IKKα/β, IκBα, and IKKα were determined by Western blotting. B and C, IKK activity was determined by kinase assay (KA), and the level of immunoprecipitated IKKα was determined by Western blotting as a loading control. D, IKK activity was quantified by densitometry (*, p < 0.05). Error bars indicate S.E.

FIGURE 4.

PP2A activity is inhibited by oxidants or OA, leading to prolonged IKK phosphorylation. A, untreated A549 cells were lysed, and cell lysates were left untreated (control, c) or were treated with 500 μm PN or with 100 nm OA for 30 min. PP2A activity was determined using a Ser/Thr phosphatase assay specific for PP2A. B, A549 cells were unstimulated (control, c) or were stimulated with 500 μm PN or 100 nm OA for 30–90 min. Cells were lysed, and PP2A activity was determined using the PP2A assay (*, p < 0.05). Error bars indicate S.E. C, A549 cells were treated with10 ng/ml TNFα for 10 min followed by PBS-glucose buffer (PBS post-TNFα), PN 500 μm (PN post-TNFα), or 100 nm OA (OA post-TNFα) for the indicated times. Phosphorylated IKKα/β was determined by Western blotting.

FIGURE 5.

Preexposure to oxidants prevents NF-κB activation by TNFα. A and B, A549 cells were left untreated (control, c) or were stimulated with 0–500 μm PN for 1 h followed by 10 ng/ml TNFα for 30 min. IκBα, nuclear p65, and α-tubulin were determined by Western blotting. C, A549 cells were left untreated (control, c) or were treated with decomposed PN (DP), 500 μm PN, or with 0–500 μm PN for 1 h followed by 10 ng/ml TNFα for 30 min. NF-κB-DNA binding activity was determined by EMSA. D, A549 cells were untreated (control, c) or treated with 500 μm PN or TNFα 10 ng/ml for 10 min or with 500 μm PN for 1 h followed by 10 ng/ml TNFα for 10 min. IKK activity was determined by kinase assay (KA), quantified by densitometry (*, p < 0.05). E, A549 cells were unstimulated or stimulated with TNFα for 4 h or with 500 μm PN for 1 h followed by TNFα for 4 h. Luciferase activity was determined in cell lysates at the end of stimulation (*, p < 0.05). Error bars indicate S.E.

FIGURE 6.

Regulation of NF-κB by oxidants is independent of the nature of the immune stimulus. A, A549 cells were unstimulated (control, c) or were stimulated with 1 ng/ml IL-1β for 30 min in the presence of a pretreatment with PBS buffer (PBS pre-IL-1) or 500 μm PN (PN pre-IL-1) for 1 h, or in the presence of a posttreatment with PBS (PBS post-IL-1) or PN (PN post-IL-1) for 1 h. NF-κB-DNA binding activity was determined by EMSA. B, upper panel, luciferase activity in A549 cells either unstimulated (c) or stimulated with PBS buffer (PBS pre-IL-1) or 500 μm PN (PN pre-IL-1) for 1 h followed by 1 ng/ml IL-1β for 4 h (*, p < 0.05) is shown. Lower panel, luciferase activity in unstimulated A549 cells (c) or in cells treated with 1 ng/ml IL-1β followed by PBS (PBS post-IL-1) or 500 μm PN (PN post-IL-1) for 4 h is shown. *, p < 0.05. Error bars indicate S.E.

FIGURE 7.

Proposed scheme of interactions among oxidants, IKK, and the NF-κB pathway. A, baseline. Randomly phosphorylated IKK is dephosphorylated by PP2A, preventing NF-κB activation. B, TNFα. IKK phosphorylation is transient, due to PP2A activity, leading to transient NF-κB activation. C, oxidant stress. Inactivation of PP2A promotes a slow accumulation of phosphorylated IKK, resulting in low grade NF-κB activation. D, oxidant stress before TNFα. IKK inhibition by oxidants prevents NF-κB activation. E, oxidant stress after TNFα. IKK phosphorylation by TNFα is not removed due to PP2A inhibition, resulting in prolonged, amplified signal.