Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells.

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5'-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a 'calm down' signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001.

Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement.

We investigated the role of prostaglandin D2 (PGD2) signaling in acute lung injury (ALI), focusing on its producer-effector interaction in vivo. Administration of endotoxin increased edema and neutrophil infiltration in the WT mouse lung. Gene disruption of hematopoietic PGD synthase (H-PGDS) aggravated all of the symptoms. Experiments involving bone marrow transplantation between WT and H-PGDS-deficient mice showed that PGD2 derived from alveolar nonhematopoietic lineage cells (i.e., endothelial cells and epithelial cells) promotes vascular barrier function during the early phase (day 1), whereas neutrophil-derived PGD2 attenuates its own infiltration and cytokine expression during the later phase (day 3) of ALI. Treatment with either an agonist to the PGD2 receptor, DP, or a degradation product of PGD2, 15-deoxy-Δ(12,14)-PGJ2, exerted a therapeutic action against ALI. Data obtained from bone marrow transplantation between WT and DP-deficient mice suggest that the DP signal in alveolar endothelial cells is crucial for the anti-inflammatory reactions of PGD2. In vitro, DP agonism directly enhanced endothelial barrier formation, and 15-deoxy-Δ(12,14)-PGJ2 attenuated both neutrophil migration and cytokine expression. These observations indicate that the PGD2 signaling between alveolar endothelial/epithelial cells and infiltrating neutrophils provides anti-inflammatory effects in ALI, and suggest the therapeutic potential of these signaling enhancements.

Activation of adipogenesis by lipocalin-type prostaglandin D synthase-generated Δ¹²-PGJ2 acting through PPARγ-dependent and independent pathways.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS)-produced PGD(2) accelerates adipogenesis. In this study, we investigated the molecular mechanism of PGD(2)-mediated activation of adipogenesis in mouse adipocytic 3T3-L1 cells. LC/MS analysis showed that Δ(12)-PGJ(2), one of the PGD(2) metabolites, was predominantly produced in the differentiated 3T3-L1 cells. Δ(12)-PGJ(2) enhanced the expression of adipogenic genes in a Δ(12)-PGJ(2)-concentration-dependent manner. Suppression of the expression of the adipogenic genes by L-PGDS siRNA or AT-56, an L-PGDS inhibitor, was cleared by the addition of Δ(12)-PGJ(2). Moreover, the production of adiponectin and leptin was increased by treatment with Δ(12)-PGJ(2). Furthermore, the results of a mammalian two-hybrid assay demonstrated that Δ(12)-PGJ(2) enhanced the PPARγ-mediated transcription activity. However, Δ(12)-PGJ(2)-activated expression of adipogenic genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase was inhibited only at 38% and 42%, respectively, by treatment with GW9662, a PPARγ antagonist in 3T3-L1 cells, although Troglitazone-mediated activation of the expression of these adipogenic genes was completely suppressed by GW9662, suggesting the existence of a PPARγ-independent mechanism for Δ(12)-PGJ(2)-activated adipogenesis. These results, taken together, indicate that Δ(12)-PGJ(2) is a dominant metabolite of L-PGDS-produced PGD(2) during adipogenesis and acts as an activator for adipogenesis through both PPARγ-dependent and -independent mechanisms in 3T3-L1 cells.

Tetranor PGDM analyses for the amyotrophic lateral sclerosis: positive and simple diagnosis and evaluation of drug effect.

Amyotrophic lateral sclerosis (ALS) is a late-onset, progressive motor neuronal degenerative disease occurring as sporadically and as a familial disorder. The patients with ALS typically become progressively paralyzed and develop respiratory failure that eventually leads to death within 3-5years. For this disease, there is no effective diagnostic method and also drug. This report describes a simple and useful diagnostic biomarker for ALS. Our findings suggest that the combination analysis of a metabolite of prostaglandin D2, 11,15-dioxo-9-hydroxy-,2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM and tPGDM) with creatinine is the diagnostic approach for ALS with high accuracy. tPGDM has the potential to be an important diagnostic tool in the pre-symptomatic stages and progression evaluation of ALS, and also to be a biomarker for the evaluation of drug effect.

Inhibition of prostaglandin D synthase suppresses muscular necrosis.

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.

Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase.

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Prostaglandin F2alpha synthase activities of aldo-keto reductase 1B1, 1B3 and 1B7.

Here, we show that three enzymes belonging to the 1B group of the aldo-keto reductase (AKR) superfamily, i.e., human placental aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction of prostaglandin (PG) H(2), a common intermediate of various prostanoids, to form PGF(2alpha) in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH(2) (K(m) = 1.9, 9.3 and 3.8 microM, respectively) and V(max) values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF(2alpha) synthase (AKR1C3; 18 microM and 4 nmol/min/mg protein, respectively). The PGF(2alpha) synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (K(i) = 3.6 and 0.26 microM, respectively) and sorbinil (K(i) = 21.7 and 0.89 microM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to these inhibitors (K(i) = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members and PGF(2alpha) as mediators of physiological and pathological processes in mammalian organisms.

A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of molecules essential in parasite metabolism of the anti-chagasic drugs are fundamental for the development of new strategies for rational drug design and/or the improvement of the current chemotherapy. While searching for a prostaglandin (PG) F(2alpha) synthase homologue, we have identified a novel "old yellow enzyme" from T. cruzi (TcOYE), cloned its cDNA, and overexpressed the recombinant enzyme. Here, we show that TcOYE reduced 9,11-endoperoxide PGH(2) to PGF(2alpha) as well as a variety of trypanocidal drugs. By electron spin resonance experiments, we found that TcOYE specifically catalyzed one-electron reduction of menadione and beta-lapachone to semiquinone-free radicals with concomitant generation of superoxide radical anions, while catalyzing solely the two-electron reduction of nifurtimox and 4-nitroquinoline-N-oxide drugs without free radical production. Interestingly, immunoprecipitation experiments revealed that anti-TcOYE polyclonal antibody abolished major reductase activities of the lysates toward these drugs, identifying TcOYE as a key drug-metabolizing enzyme by which quinone drugs have their mechanism of action.