Koichiro Takeshige

Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ

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.

1-Methyl-4-phenylpyridinium (MPP+) induces NADH-dependent superoxide formation and enhances NADH-dependent lipid peroxidation in bovine heart submitochondrial particles

We studied the effects of 1-methyl-4-phenylpyridinium (MPP+), a metabolite of a parkinsonism-inducing drug, on the superoxide formation and the lipid peroxidation in bovine heart submitochondrial particles. The NADH-supported formation of superoxide radicals was induced by MPP+ at the concentration which is considered to exist in mitochondria of dopamine neurons. The formation increased as the NADH-ubiquinone reductase activity was inhibited by MPP+. The NADH-supported lipid peroxidation by the particles in the presence of ADP-Fe3+ chelate was also enhanced by MPP+ at similar concentrations. The formation was inhibited by succinate and the reduction of endogenous ubiquinone seems to be related to the inhibition. A possibility was discussed that the formation of superoxide anions and the lipid peroxidation may contribute in the cytotoxicity of the drug.

TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-κB in lipopolysaccharide-stimulated macrophages

Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor‐κB (NF‐κB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor‐β activated kinase 1 (TGF‐TAK1), a mitogen‐activated protein kinase kinase kinase (MAPKKK), in the LPS‐induced signaling cascade. A kinase‐negative mutant of TAK1 inhibited the LPS‐induced NF‐κB activation both in a macrophage‐like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll‐like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS‐induced signaling pathway.

Diacylglycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide-generation from human neutrophils

1-Oleoyl-2-acetyl-glycerol which activates Ca2+-activated phospholipid-dependent protein kinase, induced the superoxide-production of human neutrophils, while other diacylglycerols did not. The induction was independent of extracellular calcium and did not accompany the increase of the intracellular free calcium. The superoxide-release by the diacylglycerol was inhibited by retinal, the inhibitor of the protein kinase. The diacylglycerol stimulated the phosphorylation of at least 4 proteins in intact neutrophils, the phosphorylation of which was stimulated by phorbol 12-myristate 13-acetate, the activator of the protein kinase. These observations indicate the possible involvement of the kinase in the induction process.

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.

Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing the superoxide (O2-) generation of human neutrophils, but it enhanced the O2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) about 2-fold when the cells had been preincubated with ET-1 for 10 min at 37 degrees C. The concentration at which ET-1 was 50% effective was 1 x 10(-10) M, and the maximal effect was obtained at 1 x 10(-8) M. The enhancement was observed over the range of the effective concentrations of FMLP (10(-8)-10(-6) M). ET-1 did not promote the mobilization of intracellular calcium ions and the enhancing effect of ET-1 did not change when calcium ions were depleted. These findings indicate that ET-1 is a potent modulator of human neutrophils and may thus contribute to the inflammatory process.

Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.

Stimulus-specific Induction of a Novel Nuclear Factor-κB Regulator, IκB-ζ, via Toll/Interleukin-1 Receptor Is Mediated by mRNA Stabilization

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

Inhibition of Superoxide Production and Ca2+ Mobilization in Human Neutrophils by Halothane, Enflurane, and Isoflurane

The inhibitory effects of three inhalation anesthetics, i.e., halothane, enflurane, and isoflurane, on superoxide production and the intracellular mobilization of calcium in human neutrophils were studied. The superoxide production induced by N-formyl-methionylleucyl-phcnylalanine (FMLP) was inhibited by the anesthetics, but the binding of FML|3H|P to the cells and the superoxide-forming NADPH oxidasc of the phagocytic vesicles were not inhibited. The inhibition of the cellular superoxide production was partially reversed by the addition of a calcium ionophore, A23187. The increase in intracellular free calcium monitored by a calcium-sensitive fluorescent probe, quin-2 and the release of calcium from hydrophobic environment monitored by chlortetracycline were inhibited dose dependently by the anesthetics. These observations suggest that decreased mobilization of intracellular Ca2+ is one of the mechanisms by which the anesthetics inhibited the superoxide production of human neutrophils stimulated by FMLP.

In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5′ ends of true replicate from those of abortive ones, we examined the 5′ ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5′ ends. Treatment of DNA with RNase H frees 5′ ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.