Threonine 11 of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase may interact with NAD(+) during catalysis

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.     

Thiazolidinediones as a novel class of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of prostaglandins and other nonprostanoid compounds. This enzyme was found to be dramatically induced in hormone-responsive human prostate cancer cells by androgens [M. Tong, and H. H. Tai, 2000, Biochem. Biophys. Res. Commun. 276, 77-81] and could be involved in prostate tumorigenesis. Inhibitors of this enzyme may be of value in determining the utility of these compounds in cancer chemoprevention. Previously, ciglitazone, an antidiabetic thiazolidinedione, was found to be a potent inhibitor of 15-PGDH. Structure-activity analysis of available thiazolidinediones indicated that the nature of the moiety linking to phenyl ring through ether linkage and benzylidene configuration play important roles in inhibitory potency. Furthermore, N-methylation of 2,4-thiazolidinedione abolished the inhibitory activity. A series of benzylidene thiazolidinediones with varied ring structure and methylene bridge to phenyl ring through ether linkage were synthesized and assayed for inhibitory activity. It was found that compound CT-8 (5-[4-(cyclohexylethoxy)benzylidene]-2,4-thiazolidinedione) was the most potent inhibitor effective at nanomolar range. Kinetic studies revealed that inhibition by this compound was noncompetitive with respect to NAD(+) and uncompetitive with respect to prostaglandin E(2), indicating that the inhibitor interacts with the enzyme at a site distinct from the substrate binding site. This regulatory site appears to overlap with the activator site occupied by imipramine since activation of the enzyme by this activator is competitively inhibited by compound CT-8.     

Cloning and sequence analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.     

Monoclonal antibodies that inhibit the enzyme activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

Three hybridoma cell lines secreting antibodies against human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG2b from these cell lines recognized a distinct band of Mr 28,000 on SDS/PAGE from the purified enzyme as well as a band of Mr 56,000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent Km for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and structural characterization of placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase. The primary structure reveals the enzyme to belong to the short-chain alcohol dehydrogenase family

Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Spectrofluorometric assay of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity

A simple, rapid, and sensitive spectrofluorometric assay for 15-hydroxyprostaglandin dehydrogenase activity was developed in which the rate of production of NADH was monitored. The cytosolic fraction prepared from human placental tissue was employed as the enzyme source. The assay was conducted at pH 9.5 since 15-ketoprostaglandin Δ¹³-reductase and NADH oxidase activities were inhibited at this pH, thereby minimizing the interference of the reactions catalyzed by these enzymes in the assay of prostaglandin dehydrogenase activity.     

NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney.

Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.     

15-hydroxyprostaglandin dehydrogenase (15-PGDH) and lung cancer

15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-linked oxidation of 15 (S)-hydroxyl group of prostaglandins and lipoxins and is the key enzyme responsible for the biological inactivation of these eicosanoids. The enzyme was found to be under-expressed as opposed to cyclooxygenase-2 (COX-2) being over-expressed in lung and other tumors. A549 human lung adenocarcinoma cells were used as a model system to study the role of 15-PGDH in lung tumorigenesis. Up-regulation of COX-2 expression by pro-inflammatory cytokines in A549 cells was accompanied by a down-regulation of 15-PGDH expression. Over-expression of COX-2 but not COX-1 by adenoviral-mediated approach also attenuated 15-PGDH expression. Similarly, over-expression of 15-PGDH by the same strategy inhibited IL-1beta-induced COX-2 expression. It appears that the expression of COX-2 and 15-PGDH is regulated reciprocally. Adenoviral-mediated transient over-expression of 15-PGDH in A549 cells resulted in apoptosis. Xenograft studies in nude mice also showed tumor suppression with cells transiently over-expressing 15-PGDH. However, cells stably over-expressing 15-PGDH generated tumors faster than those control cells. Examination of different clones of A549 cells stably expressing different levels of 15-PGDH indicated that the levels of 15-PGDH expression correlated positively with those of mesenchymal markers, and negatively with those of epithelial markers. It appears that the stable expression of 15-PGDH induces epithelial-mesenchymal transition (EMT) which may account for the tumor promotion in xenograft studies. A number of anti-cancer agents, such as transforming growth factor-beta1 (TGF-beta1), glucocorticoids and some histone deacetylase inhibitors were found to induce 15-PGDH expression. These results suggest that tumor suppressive action of these agents may, in part, be related to their ability to induce 15-PGDH expression.     

Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase

An NAD-linked 15-hydroxyprostaglandin dehydrogenase has been purified 13, 100-fold from human placental tissue. The specific activity of the purified enzyme ranges from 6900 to 8300 mU/mg protein depending on the method used to determine the protein concentration. On discontinous electrophoresis in sodium dodecyl sulfate more than 95% of the protein migrates as a single band; its estimated molecular weight is 25.5–26.0 kDa. This is half the value obtained when the molecular weight is estimated under non-denaturing conditions and suggests that the enzyme is composed of two identical or nearly identical subunits.     

Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase

An NAD-linked 15-hydroxyprostaglandin dehydrogenase has been purified 13,100-fold from human placental tissue. The specific activity of the purified enzyme ranges from 6900 to 8300 mU/mg protein depending on the method used to determine the protein concentration. On discontinuous electrophoresis in sodium dodecyl sulfate more than 95% of the protein migrates as a single band; its estimated molecular weight is 25.5-26.0 kDa. This is half the value obtained when the molecular weight is estimated under non-denaturing conditions and suggests that the enzyme is composed of two identical or nearly identical subunits.     

Inhibition of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) by cyclooxygenase inhibitors and chemopreventive agents

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. This enzyme is markedly induced by androgens in hormone-sensitive human prostate cancer cells (Tong M., Tai H. H. Biochem Biophys Res Commun 2000; 276: 77-81) and may be involved in tumorigenesis. Inhibition of this enzyme may be of value in anticancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenases (COXs) have been shown to be chemopreventive in epidemiological and animal-model studies. However, chemoprevention by these drugs may not be directly related to their inhibition of COXs. Other targets may be also involved in their chemopreventive activity. We have examined a variety of NSAIDs including COX-2 selective inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists and phytophenolic compounds which have been shown to be chemopreventive for their effect on 15-PGDH. It was found that most of these compounds were potent inhibitors of 15-PGDH. Among these compounds, ciglitazone appeared to be the most powerful inhibitor (IC(50)=2.7 microM). Inhibition by ciglitazone was non-competitive with respect to NAD(+) and uncompetitive with respect to PGE(2).     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

Inhibition of 15-hydroxyprostaglandin dehydrogenase by antiallergic agents

Glucagon addition to isolated rat hepatocytes increases the level of Cyclic AMP inside the cells and the activity of the enzyme phenylalanine hydroxylase. These effects of glucagon are time and dose dependent and are detectable at hormone concentration as low as 0.02nM. The glucagon concentrations causing half-maximal increases in Cyclic AMP production and phenylalanine hydroxylase activity are 0.2nM and 0.1 nM respectively. When hepatocytes are incubated with norepinephrine or the ionophore A23187, at concentrations between 1 nM and 10 μM, a slight increase in enzyme activity is seen only at the highest dose of either drug. The effect of norepinephrine can be completely antagonized by 20 μM propranolol but not by 20 μM ergocryptine. These results suggest that the activity of phenylalanine hydroxylase can be hormonally regulated, $\text{in vivo}$, through a phosphorylation mechanism catalyzed by a Cyclic AMP-dependent protein kinase.     

Inhibition of 15-hydroxyprostaglandin dehydrogenase by papaverine.

Papaverine was found to inhibit NAD⁺-linked 15-hydroxyprostaglandin dehydrogenase partially purified from guinea pig lung. The inhibition was noncompetitive with prostaglandin E₂, uncompetitive with NAD⁺, and reversible. The Ki was calculated to be 26 μM. Papaverine also inhibited the enzyme from swine lung, chicken and dog heart, and rat and dog kidney. The inhibitory effects of papaverine on the 15-hydroxyprostaglandin dehydrogenase were compared with those on cyclic AMP phosphodiesterases in these tissues.     

胃癌组织中15-PGDH的表达特征及其临床意义

目的:探讨15-羟基前列腺素脱氢酶(15-PGDH)在胃癌组织和癌旁正常组织中的表达及其与临床病理特征的关系,初步评价15-PGDH在胃癌发生、发展中的作用及其意义。方法:随机收集60例胃癌手术病人的癌组织及相应癌旁正常组织,应用免疫组织化学检测15-PGDH表达特征并进行评分,进一步分析15-PGDH的表达与胃癌临床病理参数的关系。结果:胃癌组织中15-PGDH的表达水平显著降低甚至缺失(p<0.01)。结合胃癌的临床病理学特征统计分析表明,15-PGDH在低分化胃癌中明显低于高分化胃癌中的表达(p<0.05);15-PGDH在TNMⅢ期和Ⅳ期患者的胃癌组织中的染色明显低于在Ⅰ期和Ⅱ期组织中的表达(p<0.05);存在淋巴结转移的胃癌组织中15-PGDH表达显著低于无淋巴结转移胃癌组织中的表达(p<0.01)。结论:胃癌中15-PGDH的表达减少或缺失可能是胃癌发生、发展及浸润转移的重要机制之一,15-PGDH在胃癌中可能扮演着抑癌基因的角色。