Nanae Nagata

Prostaglandin F2α 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.

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2α Synthase

Prostaglandin (PG) F2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF2α suppressed adipocyte differentiation by acting through FP receptors.

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 μm)-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 PGH2 (Km = 14 μm) with a Ki value of 75 μm 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 PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 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 PGE2 and PGF2α 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.

De novo synthesis, uptake and proteolytic processing of lipocalin-type prostaglandin D synthase, β-trace, in the kidneys

Lipocalin‐type prostaglandin D synthase (L‐PGDS) is a multifunctional protein that produces prostaglandin D2 and binds and transports various lipophilic substances after secretion into various body fluids as β‐trace. L‐PGDS has been proposed to be a useful diagnostic marker for renal injury associated with diabetes or hypertension, because the urinary and plasma concentrations are increased in patients with these diseases. However, it remains unclear whether urinary L‐PGDS is synthesized de novo in the kidney or taken up from the blood circulation. In crude extracts of monkey kidney and human urine, we found L‐PGDS with its original N‐terminal sequence starting from Ala23 after the signal sequence, and also its N‐terminal‐truncated products starting from Gln31 and Phe34. In situ hybridization and immunohistochemical staining with monoclonal antibody 5C11, which recognized the amino‐terminal Ala23–Val28 loop of L‐PGDS, revealed that both the mRNA and the intact form of L‐PGDS were localized in the cells of Henle’s loop and the glomeruli of the kidney, indicating that L‐PGDS is synthesized de novo in these tissues. However, truncated forms of L‐PGDS were found in the lysosomes of tubular cells, as visualized by immunostaining with 10A5, another monoclonal antibody, which recognized the three‐turn α‐helix between Arg156 and Thr173. These results suggest that L‐PGDS is taken up by tubular cells and actively degraded within their lysosomes to produce the N‐terminal‐truncated form.

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.

Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo‐keto reductase 1B1 – prostaglandin D2 synthase activity in the absence of NADP(H)

Aldo‐keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F2α (PGF2α) synthases, respectively, which catalyze the NADPH‐dependent reduction of PGH2, a common intermediate of various prostanoids, to form PGF2α. In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH2 to PGD2 in the absence of NADPH or NADP+. Both PGD2 and PGF2α synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP+ or after heat treatment of these enzymes at 100 °C for 5 min. The Km, Vmax, pK and optimum pH values of the PGD2 synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μm, 151 and 57 nmol·min−1·(mg protein)−1, 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF2α synthase activity were 29 and 33 μm, 169 and 240 nmol·min−1·(mg protein)−1, 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site‐directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD2 and PGF2α synthase activities. These results, together with molecular docking simulation of PGH2 to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD2 and PGF2α in the absence of NADPH or NADP+ and in the presence of NADPH, respectively.

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.

PGD2-CRTH2 Pathway Promotes Tubulointerstitial Fibrosis

Urinary excretion of lipocalin-type PGD(2) synthase (L-PGDS), which converts PG H(2) to PGD(2), increases in early diabetic nephropathy. In addition, L-PGDS expression in the tubular epithelium increases in adriamycin-induced nephropathy, suggesting that locally produced L-PGDS may promote the development of CKD. In this study, we found that L-PGDS-derived PGD(2) contributes to the progression of renal fibrosis via CRTH2-mediated activation of Th2 lymphocytes. In a mouse model, the tubular epithelium synthesized L-PGDS de novo after unilateral ureteral obstruction (UUO). L-PGDS-knockout mice and CRTH2-knockout mice both exhibited less renal fibrosis, reduced infiltration of Th2 lymphocytes into the cortex, and decreased production of the Th2 cytokines IL-4 and IL-13. Furthermore, oral administration of a CRTH2 antagonist, beginning 3 days after UUO, suppressed the progression of renal fibrosis. Ablation of IL-4 and IL-13 also ameliorated renal fibrosis in the UUO kidney. Taken together, these data suggest that blocking the activation of CRTH2 by PGD(2) might be a strategy to slow the progression of renal fibrosis in CKD.

Orally administered rubiscolin-6, a δ opioid peptide derived from Rubisco, stimulates food intake via leptomeningeal lipocallin-type prostaglandin D synthase in mice

SCOPE We found that rubiscolin-6, a δ opioid agonist peptide derived from d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a major protein of green leaves, stimulates food intake after oral administration in mice. We therefore investigated its mechanism. METHODS AND RESULTS Orexigenic activity after oral administration of rubiscolin-6 was blocked by central administration of naltrindole, an antagonist for δ opioid receptor, suggesting that orally administered rubiscolin-6 stimulates food intake via central δ opioid receptor activation. The orexigenic activity of rubiscolin-6 was inhibited by celecoxib, a cyclooxygenase (COX)-2 inhibitor. The hypothalamic mRNA expression of COX-2 and lipocallin-type (L) prostaglandin D synthase (PGDS) was elevated in response to rubiscolin-6 administration. Rubiscolin-6 stimulated food intake in wild-type and hematopoietic (H)-PGDS knockout (KO), but not L-PGDS KO mice. Interestingly, rubiscolin-6 stimulated food intake in L-PGDS(flox) /Nescre mice, which were deficient in L-PGDS in the brain parenchyma, but not leptomeninges. The orexigenic effect of rubiscolin-6 was abolished by genetic deletion of DP(1) receptor for PGD(2) , and by MK0524 or BIBO3304, an antagonist of DP(1) receptor or of Y(1) receptor for neuropeptide Y, respectively. CONCLUSION Orally administered rubiscolin-6 may stimulate food intake through COX-2 and leptomeningeal L-PGDS, followed by DP(1) and Y(1) receptors, downstream of the central δ opioid receptor.

Increased expression and cellular localization of lipocalin-type prostaglandin D synthase in Helicobacter pylori-induced gastritis

Immunological responses in the host can result in different disease outcomes of Helicobacter pylori‐induced gastritis. Prostaglandin E2 derived from cyclooxygenase (COX) and prostaglandin E synthase contribute to gastric protection. Recently, prostaglandin D2 was shown to be involved in host immunity by chemotactic activity through chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2), but its role in H. pylori‐induced gastritis has not been clarified. We determined the expression levels of mRNAs for haematopoietic PGD synthase (H‐PGDS) and lipocalin‐type PGDS (L‐PGDS), MIP‐1 alpha, IFN‐gamma, IL‐4, and CDX2 in H. pylori‐induced gastritis mucosa by quantitative RT‐PCR. We found that L‐PGDS was constitutively expressed in the epithelium of the glandular base. L‐PGDS, but not H‐PGDS, was induced on fibroblasts close to infiltrating cells in the H. pylori‐infected gastric mucosa. These fibroblasts co‐expressed COX‐2. The level of L‐PGDS mRNA expression decreased as gastritis became more severe. In most of the H. pylori‐infected gastric mucosa, CCR5(+) cells had more actively infiltrated than had CRTH2(+) cells. However, the expression level of IFN‐gamma was lower in the mucosa of the CRTH2(+) cells‐dominantly infiltrating group than that of the less CRTH2‐infiltrating group. Exogenously added PGD2 decreased the H. pylori‐induced expression of IFN‐gamma in peripheral blood mononuclear cells in vitro. The data suggest that PGD2 derived from the gastric mucosa and fibroblasts plays protective roles against inflammatory changes in H. pylori‐induced gastritis. Copyright