Kikuko Watanabe

Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.

Close kinship of human 20alpha-hydroxysteroid dehydrogenase gene with three aldo-keto reductase genes.

20α‐Hydroxysteroid dehydrogenase (HSD) is a member of the aldo‐keto reductase (AKR) superfamily and catalyses the reaction of progesterone to the inactive form 20α‐hydroxyprogesterone. Progesterone plays an important role in the maintenance of pregnancy, and, in rodents, plasma progesterone levels decrease abruptly just before parturition. The induction of 20α‐HSD is thought to be responsible for the decrease in plasma progesterone at term. High homology between human 20α‐HSD [AKR 1C1] cDNA with other AKRs had caused difficulty in gene isolation and expression analysis. Thus, the metabolism of progesterone in the human reproductive system remained unclear.

Prostaglandin F synthase

Prostaglandin (PG) F2 is synthesized via three pathways from PGE2, PGD2, or PGH2 by PGE 9-ketoreductase, PGD 11-ketoreductase, or PGH 9-, 11-endoperoxide reductase, respectively. The enzymological and molecular biological properties of these enzymes have been reported in work over the last 30 years. Here, these three pathways of PGF synthesis by these enzymes are reviewed, and the physiological roles of the enzymes are discussed.

cDNA cloning, expression and characterization of human prostaglandin F synthase1

A cDNA clone of prostaglandin F synthase (PGFS) was isolated from human lung by using cDNA of bovine lung‐type PGFS as a probe and its protein expressed in Escherichia coli was purified to apparent homogeneity. The human PGFS catalyzed the reduction of prostaglandin (PG) D2, PGH2 and phenanthrenequinone (PQ), and the oxidation of 9α,11β‐PGF2 to PGD2. The k cat/K m values for PGD2 and 9α,11β‐PGF2 were 21 000 and 1800 min−1 mM−1, respectively, indicating that the catalytic efficiency for PGD2 and 9α,11β‐PGF2 was the highest among the various substrates, except for PQ. The PGFS activity in the cytosol of human lung was completely absorbed with anti‐human PGFS antiserum. Moreover, mRNA of PGFS was expressed in peripheral blood lymphocytes and the expression in lymphocytes was markedly suppressed by the T cell mitogen concanavalin A. These results support the notion that human PGFS plays an important role in the pathogenesis of allergic diseases such as asthma.

PURIFICATION AND CHARACTERIZATION OF MEMBRANE-BOUND PROSTAGLANDIN E SYNTHASE FROM BOVINE HEART

Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) was 24 microM. The molecular weight of the enzyme was about 31000 on SDS-polyacrylamide gel electrophoresis and was about 60000 by gel filtration. The enzyme was separated from glutathione (GSH) S-transferase by DEAE-Toyopearl column chromatography, and did not exhibit any GSH S-transferase activity towards four different substrates. The purified enzyme was active in the absence of GSH, but it was activated by various SH-reducing reagents including dithiothreitol, GSH, or beta-mercaptoethanol. This is the first reported purification of membrane-bound PGE synthase to apparent homogeneity.

Expression of Key Prostaglandin Synthases in Equine Endometrium During Late Diestrus and Early Pregnancy

Abstract Luteolysis in domestic species is mediated by the release of luteolytic pulses of prostaglandin (PG) F2α by the uterus at the end of diestrus, which must be suppressed by the conceptus to permit maternal recognition of pregnancy. In many species, including the horse, both the conceptus and the endometrium also synthesize PGE2, which may antagonize PGF2α by playing a luteotropic and/or antiluteolytic role. While the release of PGE2 and PGF2α by the equine endometrium in late diestrus and early pregnancy has been previously studied, the underlying prostaglandin synthase gene regulatory mechanisms remain poorly defined. To resolve this issue, cyclooxygenase-2 (COX-2), microsomal PGE2 synthase (PGES), and PGF2α synthase (PGFS) expression were examined in a series of endometrial biopsies obtained from cycling mares on Days 10, 13, and 15 postovulation, as well as from pregnant mares on Day 15. Quantification of COX-2 expression revealed significant (P < 0.01) increases in both mRNA and protein levels at Day 15 in cycling endometrium relative to other timepoints. Importantly, the level of COX-2 expression in Day 15 pregnant endometrium was found to be comparable with that observed in Day 10 and Day 13 cycling animals, suggesting that the presence of the conceptus blocks the induction of COX-2. Immunohistochemistry demonstrated that the induction of COX-2 expression on Day 15 occurs specifically in surface epithelial cells in cycling animals only. As equine PGFS had not been previously characterized, a 1380-base pair (bp) cDNA transcript was cloned by a combination of reverse transcription-PCR techniques and found to be highly homologous to bovine liver-type PGFS. The pattern of expression observed for the terminal PG synthases was distinct from that of COX-2, as PGES and PGFS mRNA and protein levels were found to be invariant throughout the timecourse and unaffected by pregnancy. Similar to COX-2, however, the PGES and PGFS proteins were found to localize mainly to the surface epithelium. Thus, this study describes for the first time the regulation and spatial distribution of COX-2, PGES, and PGFS expression in equine endometrium in late diestrus, with a marked induction of COX-2 but not of PGES and PGFS expression in uterine epithelial cells at Day 15. Furthermore, the presence of the conceptus was shown to block the induction of COX-2 expression at Day 15, suggesting an important mechanism by which it may suppress uterine PGF2α release and prevent luteolysis during early pregnancy.

CDNA CLONING, EXPRESSION, AND MUTAGENESIS STUDY OF LIVER-TYPE PROSTAGLANDIN F SYNTHASE

Prostaglandin (PG) F synthase catalyzes the reduction of PGD2 to 9α,11β-PGF2 and that of PGH2 to PGF2α on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme (K m value of 120 μm for PGD2 (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O. (1985) J. Biol. Chem. 260, 7035–7041) and the liver-type one (K m value of 10 μm for PGD2 (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992)Arch. Biochem. Biophys. 296, 17–26)). The liver-type enzyme was presently found to consist of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with aM r of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase (DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid sequence of the liver-type PGF synthase was identical with that of bovine liver DD3. The liver-type PGF synthase was expressed in COS-7 cells, and its recombinant enzyme had almost the same properties as the native enzyme. Furthermore, to investigate the nature of catalysis and/or substrate binding of PGF synthase, we constructed and characterized various mutant enzymes as follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. Although the reductase activities toward PGH2 and phenanthrenequinone (PQ) of almost all mutants were not inactivated, the K m values of R27E, R91Q, H170C, R223L, and N306Y for PGD2 were increased from 15 to 110, 145, 75, 180, and 100 μm, respectively, indicating that Arg27, Arg91, His170, Arg223, and Asn306 are essential to give a low K m value for PGD2 of the liver-type PGF synthase and that these amino acid residues serve in the binding of PGD2. Moreover, the R223L mutant among these seven mutants especially has a profound effect on k cat for PGD2 reduction. TheK m values of R223L, K225S, and S301R for PQ were about 2–10-fold lower than the wild-type value, indicating that the amino acid residues at 223, 225 and 301 serve in the binding of PQ to the enzyme. On the other hand, the K m value of H170C for PGH2 was 8-fold lower than that of the wild type, indicating that the amino acid residue at 170 is related to the binding of PGH2 to the enzyme and that Cys170 confer high affinity for PGH2. Additionally, the 5-fold increase in k cat/K m value of the N306Y mutant for PGH2 compared with the wild-type value suggests that the amino acid at 306 plays an important role in catalytic efficiency for PGH2.

Purification and characterization of prostaglandin F synthase from bovine liver

Prostaglandin D2 11-ketoreductase activity of bovine liver was purified 340-fold to apparent homogeneity. The purified enzyme was a monomeric protein with a molecular weight of about 36 kDa, and had a broad substrate specificity for porstaglandins D1, D2, D3, and H2, and various carbonyl compounds (e.g., phenanthrenequinone and nitrobenzaldehyde, etc.). Prostaglandin D2 was reduced to 9 alpha,11 beta-prostaglandin F2 and prostaglandin H2 to prostaglandin F2 alpha with NADPH as a cofactor. Phenanthrenequinone competitively inhibited the reduction of prostaglandin D2, while it did not inhibit that of prostaglandin H2. Moreover, chloride ion stimulated the reduction of prostaglandin D2 and carbonyl compounds, while it had no effect on that of prostaglandin H2. Besides, the enzyme was inhibited by flavonoids (e.g., quercetin) that inhibit carbonyl reductase, but was not inhibited by barbital and sorbinil, which are the inhibitors of aldehyde and aldose reductases, respectively. These results indicate that the bovine liver enzyme has two different active sites, i.e., one for prostaglandin D2 and carbonyl compounds and the other for prostaglandin H2, and appears to be a kind of carbonyl reductase like bovine lung prostaglandin F synthase (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O., 1985, J. Biol. Chem. 260, 7035-7041). However, the bovine liver enzyme was different from prostaglandin F synthase of bovine lung with regard to the Km value for prostaglandin D2 (10 microM for the liver enzyme and 120 microM for the lung enzyme), the sensitivity to chloride ion (threefold greater activation for the liver enzyme) and the inhibition by CuSO4 and HgCl2 (two orders of magnitude more resistant in the case of the liver enzyme). These results suggest that the bovine liver enzyme is a subtype of bovine lung prostaglandin F synthase.

Solubilization and characterization of prostaglandin E2 binding protein from porcine cerebral cortex.

Abstract: The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS) at 4°C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time‐dependent and the equilibrium was reached at around 60 min at 37°C. By dilution of the reaction mixture, the binding site‐[3H]PGE2 complex formed after 5‐min incubation slowly dissociated, whereas that formed after 60‐min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 > PGE, × PGE2α > PGE, methyl ester > PGA2 > 13,14‐dihydro‐15‐keto‐PGE2 > PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high‐ and low‐affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion‐exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.

Regulation of prostaglandin biosynthesis by interleukin-1 in cultured bovine endometrial cells

Interleukin-1 (IL1) has been shown to be a potent stimulator of prostaglandin (PG) production in bovine endometrium. The aim of the present study was to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGE2 and PGF2alpha in response to IL1A, and the intracellular mechanisms of IL1A action. Cultured bovine epithelial and stromal cells were exposed to IL1A or IL1B (0.006-3.0 nM) for 24 h. IL1A and IL1B dose-dependently stimulated PGE2 and PGF2alpha production in the stromal cells, but not in the epithelial cells. The stimulatory effect of IL1A (0.06-3.0 nM) on PG production was greater than that of IL1B. The stimulatory actions of IL1A on PG production was augmented by supplementing arachidonic acid (AA). When the stromal cells were incubated with IL1A and inhibitors of phospholipase (PL) C or PLA2 (1 microM; anthranilic acid), only PLA2 inhibitor completely stopped the stimulatory action of IL1A on PG production. Moreover, a specific cyclooxygenase-2 (COX2) inhibitor blocked the stimulatory effect of IL1A on PG production. IL1A (0.06 nM) promoted COX2 and microsomal PGE synthase-1 (PGES1) gene and its protein expression. The expression of COX1, PGES2, PGES3, and PGF synthase (PGFS) mRNA was not affected by IL1A in the stromal cells. The overall results indicate that 1) the target of IL1A and IL1B for stimulating both PGE2 and PGF2alpha production is the stromal cells, 2) IL1A is a far more potent stimulator than IL1B on PG production in stromal cells, 3) the stimulatory effect of IL1A on PG production is mediated via the activation of PLA2 and COX2, and (4) IL1A induced PG production by increasing expressions of COX2 and PGES1 mRNAs and their proteins in bovine stromal cells.