Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation.
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The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation. (+info)
Antioxidative function and substrate specificity of NAD(P)H-dependent alkenal/one oxidoreductase. A new role for leukotriene B4 12-hydroxydehydrogenase/15-oxoprostaglandin 13-reductase.
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There are several known routes for the metabolic detoxication of alpha,beta-unsaturated aldehydes and ketones, including conjugation to glutathione and reduction and oxidation of the aldehyde to an alcohol and a carboxylic acid, respectively. In this study, we describe a fourth class of detoxication that involves the reduction of the alpha,beta-carbon=carbon double bond to a single bond. This reaction is catalyzed by NAD(P)H-dependent alkenal/one oxidoreductase (AO), an enzyme heretofore known as leukotriene B4 12-hydroxydehydrogenase, 15-oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1. AO is shown to effectively reduce cytotoxic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) (k(cat) = 4.0 x 10(3) min(-1); k(cat)/K(m) = 3.3 x 10(7) min(-1) M(-1)) and acrolein (k(cat) = 2.2 x 10(2) min(-1); k(cat)/K(m) = 1.5 x 10(6) min(-1) M(-1)) and common industrial compounds such as ethyl vinyl ketone (k(cat) = 9.6 x 10(3) min(-1); k(cat)/K(m) = 8.8 x 10(7) min(-1) M(-1)) and 15-oxoprostaglandin E1 (k(cat) = 2.4 x 10(3) min(-1); k(cat)/K(m) = 2.4 x 10(9) min(-1) M(-1)). Furthermore, transfection of human embryonic kidney cells with a rat liver AO expression vector protected these cells from challenge with HNE. The concentration of HNE at which 50% of the cells were killed after 24 h increased from approximately 15 microM in control cells to approximately 70 microM in AO-transfected cells. Overexpression of AO also completely abolished protein alkylation by HNE at all concentrations tested (up to 30 microM). Thus, we describe a novel antioxidative activity of a previously characterized bioactive lipid-metabolizing enzyme that could prove to be therapeutically or prophylactically useful due to its high catalytic rate and inducibility. (+info)
Immunohistochemical localization of guinea-pig leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase.
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We have cloned cDNA for leukotriene B4 12-hydroxydehydrogenase (LTB4 12-HD)/15-ketoprostaglandin 13-reductase (PGR) from guinea-pig liver. LTB4 12-HD catalyzes the conversion of LTB4 into 12-keto-LTB4 in the presence of NADP+, and plays an important role in inactivating LTB4. The cDNA contained an ORF of 987 bp that encodes a protein of 329 amino-acid residues with a 78% identity with porcine LTB4 12-HD. The amino acids in the putative NAD+/NADP+ binding domain are well conserved among the pig, guinea-pig, human, rat, and rabbit enzymes. The guinea-pig LTB4 12-HD (gpLTB4 12-HD) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli, which exhibited similar enzyme activities to porcine LTB4 12-HD. We examined the 15-ketoprostaglandin 13-reductase (PGR) activity of recombinant gpLTB4 12-HD, and confirmed that the Kcat of the PGR activity is higher than that of LTB4 12-HD activity by 200-fold. Northern and Western blot analyses revealed that gpLTB4 12-HD/PGR is widely expressed in guinea-pig tissues such as liver, kidney, small intestine, spleen, and stomach. We carried out immunohistochemical analyses of this enzyme in various guinea-pig tissues. Epithelial cells of calyx and collecting tubules in kidney, epithelial cells of airway, alveoli, epithelial cells in small intestine and stomach, and hepatocytes were found to express the enzyme. These findings will lead to the identification of the unrevealed roles of PGs and LTs in these tissues. (+info)
Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop.
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The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain. (+info)
Identification of a novel prostaglandin reductase reveals the involvement of prostaglandin E2 catabolism in regulation of peroxisome proliferator-activated receptor gamma activation.
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This report identifies a novel gene encoding 15-oxoprostaglandin-Delta13-reductase (PGR-2), which catalyzes the reaction converting 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. The expression of PGR-2 is up-regulated in the late phase of 3T3-L1 adipocyte differentiation and predominantly distributed in adipose tissue. Overexpression of PGR-2 in cells decreases peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent transcription and prohibits 3T3-L1 adipocyte differentiation without affecting expression of PPARgamma. Interestingly, we found that 15-keto-PGE2 can act as a ligand of PPARgamma to increase co-activator recruitment, thus activating PPARgamma-mediated transcription and enhancing adipogenesis of 3T3-L1 cells. Overexpression of 15-hydroxyprostaglandin dehydrogenase, which catalyzes the oxidation reaction of PGE2 to form 15-keto-PGE2, significantly increased PPARgamma-mediated transcription in a PGE2-dependent manner. Reciprocally, overexpression of wild-type PGR-2, but not the catalytically defective mutant, abolished the effect of 15-keto-PGE2 on PPARgamma activation. These results demonstrate a novel link between catabolism of PGE2 and regulation of ligand-induced PPARgamma activation. (+info)
Restoration of leukotriene B(4)-12-hydroxydehydrogenase/15- oxo-prostaglandin 13-reductase (LTBDH/PGR) expression inhibits lung cancer growth in vitro and in vivo.
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Inhibition of pulmonary prostaglandin metabolism by exposure of animals to oxygen or nitrogen dioxide.
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The effects of exposure of animals to 100% O2 and NO2 on the rate of prostaglandin metabolism by lung and kidney were studied in vitro. Exposure of guinea pigs to 100% O2 for 48 h inhibited the metabolism of prostaglandin F2 alpha by both NAD+- and NADP+-dependent prostaglandin dehydrogenase in lung, but had no effect on the metabolism in kidney. Succinate dehydrogenase, but not glucose 6-phosphate dehydrogenase, in guinea-pig lung was inhibited by exposure to 100% O2. Exposure to 46 p.p.m. but not 16 or 29 p.p.m. NO2 for 6 h inhibited guinea-pig lung prostaglandin dehydrogenase in vitro. The inhibition of pulmonary prostaglandin dehydrogenase by exposure to 100% O2 or to 49 p.p.m. NO2 was dependent on the duration of exposure, but returned to control values within 7 days after cessation of the exposure. The pulmonary transport system responsible for removing circulating prostaglandins from the blood was not affected by exposure to 100% O2 as measured by using the isolated perfused lung. Kinetic analysis of the inhibition of pulmonary prostaglandin dehydrogenase activity in guinea pig exposed to 100% O2 showed non-competitive inhibition with respect to both prostaglandin F2 alpha and NAD+, which suggests destruction or inactivation of the enzyme. Pulmonary prostaglandin dehydrogenase appears to be inhibited by exposure to oxidant gases, which may lead to elevated prostaglandin concentrations in the lungs or in the systemic circulation. (+info)