Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IκBζ (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IκB family member Bcl-3 (refs 1–6). Transcription of IκBζ is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IκBζ is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IκBζ-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-α. Endogenous IκBζ specifically associates with the p50 subunit of NF-κB, and is recruited to the NF-κB binding site of the IL-6 promoter on stimulation. Moreover, NF-κB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IκBζ-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IκBζ-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IκBζ occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IκBζ.

The transcription factor nuclear factor-κB (NF-κB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IκBs. We here report a new member of the IκB protein family, IκB-ζ, harboring six ankyrin repeats at its carboxyl terminus. IκB-ζ mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IκB-ζ is also observed by stimulation with interleukin-1β but not by tumor necrosis factor-α. In contrast to cytosolic IκB-α, -β, and -ε, the induced IκB-ζ localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IκB-ζ inhibits the NF-κB activity without affecting the nuclear translocation of NF-κB upon stimulation. The expressed IκB-ζ preferentially associates with the NF-κB subunit p50 rather than p65 and recombinant IκB-ζ proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IκB-ζ negatively regulates NF-κB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IκB-ζ renders cells more susceptible to apoptosis induced by tumor necrosis factor-α. The proapoptotic activity of IκB-ζ further suggests that it might be one of key regulators for inflammation and other biologically relevant processes.

Although (1→3)-β-d-glucans, which are one of major fungal cell wall components, are known to activate invertebrate innate immune systems, their activities on mammalian cells remain elusive. Here, we report their activities on mouse macrophages. Among the various (1→3)-β-d-glucans, curdlan, a linear (1→3)-β-d-glucan, although not branched β-glucans, exhibits significant activity to stimulate nuclear factor-κB in macrophages. The activity of curdlan is dramatically enhanced by pretreatment with sodium hydroxide or dimethyl sulfoxide, which disrupts multiple-stranded helices of (1→3)-β-d-glucans, and is dose-dependently inhibited by a (1→3)-β-d-glucan-binding protein and by laminarioligosaccharides with (1→3)-β-d-glucosidic linkages. Intriguingly, the activity of curdlan is also augmented by incubation with zymolyase, which releases (1→3)-β-d-glucans with a single helical structure from the glucan-networks assembled by multiple-stranded helices. The activation of macrophages culminates in the production of inducible nitric-oxide synthase, tumor necrosis factor-α, and macrophage inflammatory protein-2. Furthermore, a dominant-negative mutant of MyD88, an adaptor protein mediating signaling through the Toll-like receptor/inerleukin-1 receptor-like (TIR) domain, inhibits the activation of macrophages by curdlan. These results strongly suggest that macrophages respond to linear (1→3)-β-d-glucans, possibly released from fungal cell walls, via a receptor(s) harboring the TIR domain, such as a Toll-like receptor, to induce inflammatory reactions.

Articles in recent years have described two separate and distinct NF-κB activation pathways that result in the differential activation of p50- or p52-containing NF-κB complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-κB activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-κB pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-κB activation and gene expressionin vitroandin vivo. We also demonstrate that deregulation of the canonical NF-κB pathway in TRAF3-deficient cells results from accumulation of NF-κB-inducing kinase (NIK), the essential kinase mediating noncanonical NF-κB activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-κB activation pathways.

Proper activation of nuclear factor (NF)–κB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-κB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-κB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-κB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280–288). We demonstrate that loss of tumor necrosis factor (TNF) receptor–associated factor 3 (TRAF3) results in constitutive noncanonical NF-κB activity. Importantly, TRAF3^−/− B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-κB–inducing kinase in TRAF3^−/− cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-κB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-κB pathway and that constitutive activation of the noncanonical NF-κB pathway causes the lethal phenotype of TRAF3-deficient mice.

A proline-rich region is present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P-450s. To assess the functional significance of the proline residues in this region, we systematically altered these residues of cytochrome P450(M1) (P450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and the mutated proteins were expressed in the fission yeast Schizosaccharomyces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5–9% of the total membrane protein). The expressed wild-type P450(M1) showed a typical carbon monoxide difference spectrum of P-450 and the activity of testosterone hydroxylation, whereas all the mutated proteins constructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this region resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exchanged were more sensitive to trypsin digestion than the wild type. From these results, we propose that the proline residues in the proline-rich region are crucial for the formation of the correct conformation of microsomal P-450 molecules.