No abstract

Interleukin (IL) 6 is one of major mediators of inflammation, and IL-6 gene activation during hypoxia/reoxygenation has been implicated in the pathogenesis of ischemia/reperfusion injury. However, molecular events involved in IL-6 gene expression during hypoxia/reoxygenation remain to be identified. We have previously shown that NF-kappa B plays an essential and indispensable role in the transcriptional activation of the IL-6 gene induced by various stimuli, including IL-1 and tumor necrosis factor-alpha. We show here that hypoxia, but not reoxygenation, induces the activation of NF-kappa B through the degradation of a major inhibitor of NF-kappa B, I kappa B alpha. This hypoxia-induced NF-kappa B activation resulted in the kappa B-dependent transcriptional activation of the IL-6 gene. Interestingly, the time course of hypoxia-induced NF-kappa B activation was rather slow as compared with those of NF-kappa B activation induced by other stimuli, such as IL-1: a significant NF-kappa B activation was not observed before 1 hr of hypoxia treatment and persisted for up to 7 hr of hypoxia treatment. However, hypoxia-induced NF-kappa B activation was not inhibited by cycloheximide, which indicates that hypoxia directly triggers NF-kappa B activation. Furthermore, while hypoxia is unlikely to generate reactive oxygen intermediates, pretreatment of cells with antioxidants such as N-acetyl cysteine and alpha-tocopherol inhibited NF-kappa B activation induced by hypoxia. Thus, we discuss possible implications of these results for a postulated role of reactive oxygen intermediates in NF-kappa B activation.

The immunosuppressant FK506 activates NF-B through IB␣ degradation in nonlymphoid cells. In the present study, we analyzed mechanisms by which FK506 induces IB␣ degradation. We found that FK506 induces the degradation of both IB␣ and IB␤ and that the time courses of the FK506-induced degradation are quite different from degradation induced by interleukin 1 (IL-1). Despite this difference, FK506-induced IB␣ degradation was dependent on the N-terminal Ser-32 and Ser-36 phosphorylation sites and was mediated by proteasomes, as is the case for IL-1-induced IB␣ degradation. We further showed that FK506 induces weak and slow phosphorylation of IB␣ at Ser-32. However, unlike IL-1-induced degradation, IKK-1 and IKK-2 were not activated significantly nor was FK506-induced IB␣ degradation dependent on the N-terminal ubiquitination sites (Lys-21 and Lys-22). These results therefore indicate that FK506 and IL-1 utilize similar but distinct mechanisms to induce the phosphorylation and degradation of IB␣. Nuclear factor B (NF-B)1 is a transcription factor that plays an important role in inducing the expression of diverse cellular genes, such as for various cytokines, cell surface receptors, and acute-phase proteins. It is a heterodimer mainly composed of the p50 and RelA proteins, but there might be a considerable heterogeneity in its composition in various cell types, because of the presence of p50/RelA-related proteins (p52, c-Rel, and RelB), which share extensive homology in their N-terminal DNA-binding/dimerization regions. These proteins are now known as the NF-B/Rel/Dorsal transcription factor family, as they are also related to the Drosophila maternal morphogen gene, dorsal. An unusual feature of this family is that they exist in the cytoplasm in an inactive form complexed with a family of inhibitor proteins termed IB (IB␣, IB␤, and IB⑀). A variety of stimuli, including virus infection, bacterial lipopolysaccharides, double-stranded RNA, phorbol esters, UV radiation, oxidative stress, and inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-␣ (TNF-␣) activate NF-B through the proteolytic degradation of IB and the subsequent translocation of NF-B to the nucleus, where it activates target genes (1-3).The prototypic and best-studied of the IBs is IB␣, which is phosphorylated at its N-terminal two serine residues (Ser-32 and Ser-36) prior to degradation, when cells are exposed to appropriate NF-B activators (4 -6). This phosphorylation triggers the ligation of multiple ubiquitin molecules to nearby lysine residues (Lys-21 and Lys-22), leading to the subsequent degradation of the protein by proteasomes (6 -9). The signalinduced phosphorylation of IB␣ is therefore a critical step in NF-B activation and has been investigated intensively. Recently, two closely related IB kinases (IKKs), termed IKK-1 and IKK-2, have been identified and cloned. Both kinases directly phosphorylate Ser-32 and Ser-36 of IB␣ and their activities are stimulated by IL-1 and TNF-␣ treatment (10 -14). In addition, pp90rsk kinase...