Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IkappaBzeta (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IkappaB family member Bcl-3 (refs 1-6). Transcription of IkappaBzeta is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IkappaBzeta is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IkappaBzeta-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-alpha. Endogenous IkappaBzeta specifically associates with the p50 subunit of NF-kappaB, and is recruited to the NF-kappaB binding site of the IL-6 promoter on stimulation. Moreover, NF-kappaB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IkappaBzeta-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IkappaBzeta-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IkappaBzeta 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 IkappaBzeta.

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

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome bs58, composed of 91-kDa (gp91PbOx) and 22-kDa (p22PIox) subunits, and two cytosolic factors (p47PbOX and p67Pb°'), each containing two Src homology 3 (SH3) domains.Here we show that the region of the tandem SH3 domains of p47Phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22PhO and p67PhOx but fails to interact with a mutant p22Phox carrying a Pro-156 -* Gln substitution in a prolinerich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.During ingestion of microbes or upon stimulation with various soluble molecules, neutrophils and other phagocytic cells produce superoxide (O°), a precursor of microbicidal oxidants (1-4). The process involves activation ofthe phagocyte NADPH oxidase, dormant in resting cells, that catalyzes reduction of molecular oxygen to superoxide in conjunction with oxidation of NADPH. The significance of the NADPH oxidase in host defense is made evident by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease (CGD) whose phagocytes lack the superoxide-producing system (1-4).The active NADPH oxidase is found on the phagocyte membrane as an enzyme complex, the components ofwhich are identified as targets of genetic defects causing CGD. The one identified at an earlier stage is a phagocyte-specific membrane-integrated b-type cytochrome, cytochrome b558 (5-11), composed of 91-kDa and 22-kDa subunits (designated gp91Phox and p22PhOX, respectively). The cytochrome is now considered to be a flavocytochrome comprising an apparatus transporting electrons from NADPH via FAD and then heme to molecular oxygen (12)(13)(14)(15)(16) . In addition to these specialized factors, as a third cytosolic factor, the small GTPbinding protein p21rac (rac 1 and/or rac 2) is also involved in the system (22)(23)(24).Although the components of the NADPH oxidase are thus identified, little is known about the mechanism for their assembly leading to activation of the enzyme. Upon phagocyte stimulation, the cytosolic components translocate to the membrane where cytochrome b558 resides (25,26). Experiments using neutrophils from CGD patients have revealed that the t...

The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67 phox , another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67 phox N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly ␣-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro. Thus the TPR motifs of p67 phox are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.

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IB-is an inducible nuclear protein that interacts with nuclear factor-B (NF-B) via its carboxyl-terminal ankyrin-repeats. Previous studies using an NF-B reporter have shown that IB-inhibits the activity of NF-B. In the present study, we dissected the aminoterminal region of IB-, which shows no homology to any other proteins. Indirect immunofluorescence studies demonstrated the presence of a bipartite nuclear localization signal spanning amino acids 163-178. Using GAL4 fusion proteins, we found that internal fragments containing amino acids 329 -402 possessed intrinsic transcriptional activation activity. Interestingly, the activity was not detected in GAL4 fusion proteins of the full-length IB-. On the other hand, the GAL4-dependent transcriptional activity was generated by co-expression of the GAL4-NF-B p50 subunit fusion protein and the full-length IB-, neither of which exhibited the activity on their own. A new splicing variant, IB-(D), with a deletion of amino acids 236 -429, was found to lack transactivation activity. Forced expression of IB-, but not IB-(D), augmented interleukin-6 production, indicating the functional significance of the transactivation activity. In contrast, tumor necrosis factor-␣ production was inhibited by expression of IB-, highlighting the dual functions of this molecule. These results indicate that IB-harbors latent transcriptional activation activity, and that the activity is expressed upon interaction with the NF-B p50 subunit. In addition to the inhibitory activity on NF-B-mediated transcription, the transcriptional activation activity of IBshould be crucial for the regulation of inflammation.

We have recently identified an inducible nuclear factor-B (NF-B) regulator, IB-, which is induced by microbial ligands for Toll-like receptors such as lipopolysaccharide and the proinflammatory cytokine interleukin (IL)-1␤ but not by tumor necrosis factor (TNF)-␣. In the present study, we examined mechanisms for stimulus-specific induction of IB-. The analysis of the IB-promoter revealed an essential role for an NF-B binding sequence in transcriptional activation. The activation, however, did not account for the Toll-like receptor/IL-1 receptor-specific induction of IB-, because the promoter analysis and nuclear run-on analysis indicated that its transcription was similarly induced by TNF-␣. To examine post-transcriptional regulation, we analyzed the decay of IB-mRNA, and we found that it was specifically stabilized by lipopolysaccharide or IL-1␤ but not by TNF-␣. Furthermore, we found that costimulation with TNF-␣ and another proinflammatory cytokine, IL-17, elicited the IB-induction. Stimulation with IL-17 alone did not induce IB-but stabilized its mRNA. Therefore, IB-induction requires both NF-B activation and stimulus-specific stabilization of its mRNA. Because IB-is essential for expression of a subset of NF-B target genes, the stimulus-specific induction of IB-may be of great significance in regulation of inflammatory reactions.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.