Yoshihide Kanaoka

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2‐related kinases functioning early in the cell cycle may be targets for this phosphatase.

Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses.

The cysteinyl leukotrienes (cys-LTs) are a family of potent bioactive lipids that act through two structurally divergent G protein-coupled receptors, termed the CysLT1 and CysLT2 receptors. The cloning and characterization of these two receptors has not only reconciled findings of previous pharmacologic profiling studies of contractile tissues, but also has uncovered their expression on a wide array of circulating and tissue-dwelling leukocytes. With the development of receptor-selective reagents, as well as mice lacking critical biosynthetic enzymes, transporter proteins, and the CysLT1 receptor, diverse functions of cys-LTs and their receptors in immune and inflammatory responses have been identified. We review cys-LT biosynthesis; the molecular biology and distribution of the CysLT1 and CysLT2 receptors; the functions of cys-LTs and their receptors in the recruitment and activation of effector leukocytes and induction of adaptive immunity; and the development of fibrosis and airway remodeling in animal models of lung injury and allergic inflammation.

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice

PGD2 is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD2 in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD2, but not PGE2, substantially increased in the lungs of WT and TG mice with greater PGD2 increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-γ in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-γ than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD2 caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD2 plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.

Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells

House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.

Leukotriene E4–induced pulmonary inflammation is mediated by the P2Y12 receptor

Of the potent lipid inflammatory mediators comprising the cysteinyl leukotrienes (LTs; LTC4, LTD4, and LTE4), only LTE4 is stable and abundant in vivo. Although LTE4 shows negligible activity at the type 1 and 2 receptors for cys-LTs (CysLT1R and CysLT2R), it is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in humans with asthma. We show that the adenosine diphosphate (ADP)–reactive purinergic (P2Y12) receptor is required for LTE4-mediated pulmonary inflammation. P2Y12 receptor expression permits LTE4 -induced activation of extracellular signal-regulated kinase in Chinese hamster ovary cells and permits chemokine and prostaglandin D2 production by LAD2 cells, a human mast cell line. P2Y12 receptor expression by LAD2 cells is required for competition between radiolabeled ADP and unlabeled LTE4 but not for direct binding of LTE4, suggesting that P2Y12 complexes with another receptor to recognize LTE4. Administration of LTE4 to the airways of sensitized mice potentiates eosinophilia, goblet cell metaplasia, and expression of interleukin-13 in response to low-dose aerosolized allergen. These responses persist in mice lacking both CysLT1R and CysLT2R but not in mice lacking P2Y12 receptors. The effects of LTE4 on P2Y12 in the airway were abrogated by platelet depletion. Thus, the P2Y12 receptor is required for proinflammatory actions of the stable abundant mediator LTE4 and is a novel potential therapeutic target for asthma.

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 Å resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane α-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes

Dectin-2 expression on GM-CSF–cultured bone marrow cells is required for the generation of cysteinyl leukotrienes and Th2 cytokines in response to the house dust mite Dermatophagoides farinae in vivo.

Hematopoietic prostaglandin D synthase

The biological actions of prostaglandin (PG) D(2) include vasodilatation, bronchoconstriction, inhibition of platelet aggregation, and recruitment of inflammatory cells. Characterization of DP receptor null mice in which antigen-induced airway and inflammatory responses are attenuated and identification of CRTH2 as a novel PGD(2) receptor have shed light on the role of PGD(2) in the immune and inflammatory responses. Hematopoietic PGD synthase (H-PGDS) is a cytosolic enzyme that isomerizes PGH(2), a common precursor for all PGs and thromboxanes, to PGD(2) in a glutathione-dependent manner. H-PGDS is expressed in mast cells, antigen-presenting cells, and Th2 cells, and is the only mammalian member of the Sigma class of cytosolic glutathione S-transferases. In this review, we focus on the molecular biology of H-PGDS, the determination of its three-dimensional structure, characterization of the regulation of its gene expression, and information gleaned from transgenic animals.

Cysteinyl Leukotrienes Regulate Th2 Cell-Dependent Pulmonary Inflammation

The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C4 synthase (LTC4S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC4Snull mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC4Snull mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC4Snull mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC4Snull mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.