Activation of transcription factor NF-κB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IκBα. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-κB activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of IκBα. Indeed, mutation of Ser32 and Ser36 blocked IL-1β- or PMA-induced NF-κB activation, but had no effect on its activation by H2O2. Although IκBα was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-κB activation by H2O2, while it had no effect on activation by IL-1β or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on IκB kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole) blocks NF-κB activation. H2O2-induced IκBα phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-κB activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.

Tumor necrosis factor-␣ (TNF-␣) and lymphotoxin-␤ receptor (LT␤R) signaling both play important roles in inflammatory and immune responses through activation of NF-B. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-B induction in response to TNF-␣ and LT␤R activation. We demonstrate that LT␤R ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-␣ induces only RelA/p50 dimers. LT␤R-induced binding of RelB/p50 requires processing of p100 that is mediated by IKK␣ but is independent of IKK␤, NEMO/IKK␥, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-␣ but not LT␤ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-B pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.

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