Kaoru Hazeki

Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase

Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.

Activation of the human innate immune system by Spirulina: augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis.

Spirulina platensis is a cyanobacterial species that is surmised to potentiate the immune system leading to suppression of cancer development and viral infection. Here, we identified the molecular mechanism of the human immune potentiating capacity of Spirulina by analyzing blood cells of volunteers with pre and post oral administration of hot water extract of Spirulina. NK functions represented by IFN gamma production and cytolysis were enhanced after administration of Spirulina in >50% subjects. IFN gamma was produced in an IL-12/IL-18-dependent fashion. In vitro stimulation of blood cells with BCG cell wall skeleton (CWS) allowed more potent IL-12 p40 production in cells from volunteers given Spirulina than in cells without pre-exposure to Spirulina. As BCG-CWS serves as a ligand for Toll-like receptor (TLR) 2 and 4 to raise the maturation stage of monocytes/macrophages, Spirulina may be involved in the signaling responses through Toll in blood cells even when orally administered. These observations indicated that in humans Spirulina acts directly on myeloid lineages and either directly or indirectly on NK cells. The presence of co-operative IL-12 and IL-18 is critically important for NK-mediated IFN gamma production.

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model

Background and purpose:  TAK‐242, a novel synthetic small‐molecule, suppresses production of multiple cytokines by inhibiting Toll‐like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK‐242 and examined its therapeutic effect in a mouse sepsis model.

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase.

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.

Functional Modulation of Human Macrophages Through CD46 (Measles Virus Receptor): Production of IL-12 p40 and Nitric Oxide in Association with Recruitment of Protein-Tyrosine Phosphatase SHP-1 to CD46

Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (Mφ) by cross-linking their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs F(ab′)2 against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF-cultured Mφ, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a specific maturation stage of human Mφ.

Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2+ influx.

Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from nontreated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca2+-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils.

Opposite Effects of Wortmannin and 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one Hydrochloride on Toll-Like Receptor-Mediated Nitric Oxide Production: Negative Regulation of Nuclear Factor-κB by Phosphoinositide 3-Kinase

A number of previous studies have suggested the involvement of phosphoinositide 3-kinase (PI3K) in Toll-like receptor (TLR) signaling. However, there have also been a number of conflicting reports. The PI3K inhibitor wortmannin greatly enhanced TLR-mediated inducible nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line Raw264.7. The effect of wortmannin was common to TLR2, -3, -4, and -9 and was accompanied by activation of nuclear factor-κB and up-regulation of cytokine mRNA production. We were surprised to find that another PI3K inhibitor, LY294002, strongly suppressed the production of iNOS and cytokines. This effect of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) was based on its inhibitory effect on mRNA synthesis. Expression of dominant-negative mutants of PI3K in macrophages augmented the lipopolysaccharideinduced expression of iNOS. Introduction of a pH1 vector producing short hairpin RNA that targets a catalytic subunit of PI3K (p110β) also enhanced the TLR-mediated responses. Thus, the augmentation of TLR signals by wortmannin was mediated through the inhibition of PI3K, whereas the effect of LY294002 was not explained by its effect on PI3K. These discrepancies in the effects of pharmacological inhibitors in TLR-signaling may have caused confusion regarding the role of PI3K in innate immunity.

Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways

Toll‐like receptor (TLR)‐mediated recognition of pathogens represents one of the most important mechanisms of innate immunity. A proximal signaling event of TLR is the direct binding of an adaptor protein MyD88 to TLR and recruitment of the IL‐1R‐associated kinase (IRAK). In the present study, we examined the effect of several TLR ligands on protein tyrosine phosphorylation in rat macrophages. Macrophage‐activating lipopeptide‐2 kDa (MALP2) and lipoarabinomannan were used as activators of TLR2, while lipopolysaccharides (LPS) and lipoteichoic acid were used as TLR4 ligands. All these ligands induced tyrosine phosphorylation of proline‐rich tyrosine kinase 2 (Pyk2) and its substrate paxillin, an integrin‐associated focal adhesion adaptor protein, in the macrophages. PP2, an inhibitor of Src family tyrosine kinases, prevented the TLR‐induced phosphorylation of paxillin and Pyk2 without affecting TLR‐induced IRAK activation. MALP2 failed to induce paxillin phosphorylation in the macrophages from MyD88‐knockout mice. In contrast, the effect of LPS weakened, but was still observed even in the MyD88‐deficient cells. Thus, TLR regulate the function of paxillin in an Src family‐dependent mechanism through both MyD88‐dependent and MyD88‐independent pathways.

Enhancement of antitumor natural killer cell activation by orally administered Spirulina extract in mice

Oral administration of hot‐water extract of Spirulina, cyanobacterium Spirulina platensis, leads to augmentation of NK cytotoxicity in humans. Here, we applied to syngeneic tumor‐implant mice (C57BL/6 versus B16 melanoma) Spirulina to elucidate the mechanism of raising antitumor NK activation. A B16D8 subcell line barely expressed MHC class I but about 50% expressed Rae‐1, a ligand for NK activation receptor NKG2D. The Rae‐1‐positive population of implant B16 melanoma was effectively eliminated in the tumor mass progressed in mice. This antitumor activity was induced in parallel with IFN‐γ and abolished in mice by treatment with asialoGM‐1 but not CD8β Ab, suggesting the effector is NK cell. NK cell activation occurred in the spleen of wild‐type mice medicated with Spirulina. This Spirulina‐mediated enhanced NK activation was abrogated in MyD88 –/– mice but not in TICAM‐1 –/– mice. The NK activating properties of Spirulina depending on MyD88 were confirmed with in vitro bone marrow‐derived dendritic cells expressing TLR2/4. In D16D8 tumor challenge studies, the antitumor effect of Spirulina was abolished in MyD88 –/– mice. Hence, orally administered Spirulina enhances tumoricidal NK activation through the MyD88 pathway. Spirulina exerted a synergistic antitumor activity with BCG–cell wall skeleton, which is known to activate the MyD88 pathway via TLR2/4 with no NK enhancing activity. Spirulina and BCG–cell wall skeleton synergistically augmented IFN‐γ production and antitumor potential in the B16D8 versus C57BL/6 system. We infer from these results that NK activation by Spirulina has some advantage in combinational use with BCG–cell wall skeleton for developing adjuvant‐based antitumor immunotherapy. (Cancer Sci 2009)

Critical Roles of the p110β Subtype of Phosphoinositide 3-Kinase in Lipopolysaccharide-Induced Akt Activation and Negative Regulation of Nitrite Production in RAW 264.7 Cells

It has been suggested that PI3K participates in TLR signaling. However, identifying specific roles for individual PI3K subtypes in signaling has remained elusive. In macrophages from the p110γ−/− mouse, LPS-induced phosphorylation of Akt occurred normally despite the fact that the action of anaphylatoxin C5a was impaired markedly. In RAW 264.7 cells expressing short hairpin RNA that targets p110β, LPS-induced phosphorylation of Akt was significantly attenuated. In contrast, the LPS action was not impaired, but was rather augmented in the p110α-deficient cells. Previous pharmacologic studies have suggested that a PI3K-Akt pathway negatively regulates TLR-induced inducible NO synthase expression and cytokine production. In the p110β-deficient cells, inducible NO synthase expression and IL-12 production upon stimulation by LPS were increased, whereas LPS-induced expression of COX-2 and activation of MAPKs were unaffected. Together, the results suggest a specific function of p110β in the negative feedback regulation of TLR signaling.