Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

A comprehensive immunohistochemistry of prostaglandins F2α and E2 synthetic enzymes in rat ovary and uterus around parturition.

A comprehensive immunohistochemistry with the isoform-distinguishable antibodies against prostaglandin (PG) F2α and PGE2 biosynthetic enzymes was undertaken to identify the cellular types and enzyme isoforms in rat ovary and uterus around parturition. In general ovarian and uterine cells showed positive immunoreactions for phospholipase A2 groups 4A and 6A, but not group 2A, and cyclooxygenase (COX)-1 rather than COX-2. Their immunoreactions for PGF2α synthase and PGE2 synthase were cell type-dependently variable. The putative PGF2α and PGE2 producing cell types included, as expected, ovarian luteal cells, uterine endometrial epithelium and myometrium, and cervical connective tissue and, unexpectedly, ovarian stromal cells and basal lamina of cervical endometrium. Obtained data indicate the generation of PGF2α and PGE2 by multiple sites, which are entirely the same as established sites of actions, in parturition processes and tissue-dependent differential usage of PG biosynthetic pathway.

Deletion of the gene encoding prostamide/prostaglandin F synthase reveals an important role in regulating intraocular pressure.

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide F2α and prostaglandin F2α (PGF2α.). The importance of this enzyme, relative to the aldo-keto reductase (AKR) series, in providing functional tissue prostamide F2α levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (Fam213b / Prxl2b) was accomplished by a two exon disruption. Prostamide F2α levels in wild type (WT) and PM/PGFS knock-out (KO) mice were determined by LC/MS/MS. Deletion of Fam213b (Prxl2b) had no observed effect on behavior, appetite, or fertility. In contrast, tonometrically measured intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. No effect on pressure dependent outflow facility occurred, which is consistent with the effects of prostamide F2α and PGF2α increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids known to raise intraocular pressure, namely prostaglandin D2 (PGD2) and thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2α and prostamide F2α to constrain the influence of those prostanoids that raise intraocular pressure.

Recruitment of thioredoxin-like domains into prostaglandin synthases.

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently. We have identified two new enzymes, thioredoxin-type PGF synthase and membrane-associated PGE synthase-2, with thioredoxin-like domains. In addition, the N-terminal domain of hematopoietic PGD synthase is classified into the thioredoxin-like superfamily, based on structural similarity. The active sites of the former two enzymes have a CXXC motif, which is also critical for the thioredoxin activity. In contrast, hematopoietic PGD synthase lacks the motif, and the activity is carried out by glutathione. A phylogenetic tree of the thioredoxin-like domains suggests that they have been independently recruited into these PG synthases. We will discuss the functional meaning of the thioredoxin-like domains in the PG synthases from the viewpoint of the redox activity.

Studies on membrane-associated prostaglandin E synthase-2 with reference to production of 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT).

Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH(2) primarily to PGE(2). The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic PGE synthase, both of which require specifically glutathione. Recently, other investigators reported that their preparation of mPGE synthase-2 containing heme converted PGH(2) to 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) rather than to PGE(2) [T. Yamada, F. Takusagawa, Biochemistry 46 (2007) 8414-8424]. As we examined presently, the heme-bound enzyme expressed and purified according to their method synthesized HHT from PGH(2), but also PGE(2) in a decreased amount. Whereas the PGE synthase activity was completely lost at 50 degrees C for 5 min, the HHT synthase activity remained even at 100 degrees C for 5 min. In contrast, when the heme-bound enzyme was purified in the presence of dithiothreitol, only PGE(2) was produced, but essentially no HHT was detected. Thus, native mPGE synthase-2 enzymatically catalyzes only the conversion of PGH(2) to PGE(2), but not to HHT, and heme is not involved in this reaction.

Two pathways for prostaglandin F2 alpha synthesis by the primate periovulatory follicle.

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2 alpha levels in primate periovulatory follicles. To better understand the role of PGF2 alpha in ovulation, pathways utilized for PGF2 alpha synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34-36 h after hCG) from in vitro fertilization patients. PGF2 alpha can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24-36 h after hCG administration. Human granulosa cells converted PGD2 into 11 beta-PGF2 alpha, confirming that these cells possess AKR1C3 activity. PGF2 alpha can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0-12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [(3)H]PGE2 into [(3)H]PGF2 alpha was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2 alpha can be synthesized via two pathways during the periovulatory interval.

Molecular cloning and spatiotemporal expression of prostaglandin F synthase and microsomal prostaglandin E synthase-1 in porcine endometrium.

Endometrial prostaglandins (PGs) and the PGE2/PGF2alpha ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2alpha ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10-13, intermediate on d 14-23, and high on d 24-25). In pregnancy, expression of mPGES-1 was intermediate on d 10-11 and low on d 14-17 and then increased after d 22, reaching the maximum on d 24-25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10-13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2alpha ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.

Crystal structure and possible catalytic mechanism of microsomal prostaglandin E synthase type 2 (mPGES-2).

Prostaglandin (PG) H(2) (PGH(2)), formed from arachidonic acid, is an unstable intermediate and is converted efficiently into more stable arachidonate metabolites (PGD(2), PGE(2), and PGF(2)) by the action of three groups of enzymes. Prostaglandin E synthase catalyzes an isomerization reaction, PGH(2) to PGE(2). Microsomal prostaglandin E synthase type-2 (mPGES-2) has been crystallized with an anti-inflammatory drug indomethacin (IMN), and the complex structure has been determined at 2.6A resolution. mPGES-2 forms a dimer and is attached to lipid membrane by anchoring the N-terminal section. Two hydrophobic pockets connected to form a V shape are located in the bottom of a large cavity. IMN binds deeply in the cavity by placing the OMe-indole and chlorophenyl moieties into the V-shaped pockets, respectively, and the carboxyl group interacts with S(gamma) of C110 by forming a H-bond. A characteristic H-bond chain formation (N-H...S(gamma)-H...S(gamma)...H-N) is seen through Y107-C113-C110-F112, which apparently decreases the pK(a) of S(gamma) of C110. The geometry suggests that the S(gamma) of C110 is most likely the catalytic site of mPGES-2. A search of the RCSB Protein Data Bank suggests that IMN can fit into the PGH(2) binding site in various proteins. On the basis of the crystal structure and mutation data, a PGH(2)-bound model structure was built. PGH(2) fits well into the IMN binding site by placing the alpha and omega-chains in the V-shaped pockets, and the endoperoxide moiety interacts with S(gamma) of C110. A possible catalytic mechanism is proposed on the basis of the crystal and model structures, and an alternative catalytic mechanism is described. The fold of mPGES-2 is quite similar to those of GSH-dependent hematopoietic prostaglandin D synthase, except for the two large loop sections.

Coordinate regulation of prostaglandin metabolism for induction of parturition in mice.

Prostaglandins are essential for the initiation of parturition in mice. The peak in uterine prostaglandin F(2)(alpha) levels occurs at d 19.0 of gestation, just before the onset of labor. Our studies set out to determine the important regulatory step(s) involved in this increase of prostaglandin F(2)(alpha). We show that cytosolic phospholipase A(2) mRNA, protein, and activity do not significantly vary during mouse gestation. Rather, our studies demonstrate that cyclooxygenase-1 mRNA is abruptly induced at d 15.5 of gestation, but cyclooxygenase-1 protein levels only gradually increase throughout gestation. In contrast, cyclooxygenase-2 protein remains constant during gestation. We find that prostaglandin F synthase protein increases significantly during gestation reaching peak levels between d 15.5 and d 17.5 of gestation. We also find that the level of prostaglandin dehydrogenase, responsible for degradation of prostaglandins, decreases during late gestation. Taken together these results suggest that the regulation of prostaglandin F(2)(alpha) is a complex process involving the coordinate induction of synthetic enzymes along with a decrease in degradative enzymes involved in prostaglandin metabolism.

Membrane-associated prostaglandin E synthase-1 is required for neuropathic pain.

It is widely accepted that prostaglandin (PG) E2 is the principal pro-inflammatory prostanoid and plays an important role in inflammatory pain. However whether PGE2 is involved in neuropathic pain remains unknown. PGE2 is produced from arachidonic acid via PGH2 by at least three PGE synthases (PGES), cytosolic PGES (cPGES), and membrane-associated PGES (mPGES)-1 and -2. In the present study, to clarify the involvement of PGE2 and identify PGES mediating neuropathic pain, we applied a neuropathic pain model prepared by L5 spinal nerve transection to mPGES-1 knockout (mPGES-1-/-) mice. Whereas they retained normal nociceptive responses, mPGES-1-/- mice did not exhibit mechanical allodynia and thermal hyperalgesia over a week. These results demonstrate that PGE2 produced by mPGES-1 is involved in neuropathic pain.

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.

Accumulation of S-2-hydroxyethyl derivatives of L-cysteine and L-homocysteine by a methionine auxotroph of Escherichia coli with the addition of 2-mercaptoethanol to the culture.

We previously reported [J. Biosci. Bioeng., 94, 178-181 (2002)] that an Escherichia coli MetC-deficient mutant can accumulate L-cystathionine. When 2-mercaptoethanol was added to the culture medium during fermentation, the accumulation of L-cystathionine decreased and S-(2-hydroxyethyl)-L-cysteine and S-(2-hydroxyethyl)-L-homocysteine were accumulated.

Recent reports about enzymes related to the synthesis of prostaglandin (PG) F(2) (PGF(2α) and 9α, 11ß-PGF(2)).

Prostaglandin (PG) F(2α) is widely distributed in various organs and exhibits various biological functions, such as luteolysis, parturition, aqueous humor homeostasis, vasoconstriction, rennin secretion, pulmonary fibrosis and so on. The first enzyme reported to synthesize PGF(2) was referred to as PGF synthase belonging to the aldo-keto reductase (AKR) 1C family, and later PGF(2α) synthases were isolated from protozoans and designated as members of the AKR5A family. In 2003, AKR1B5, which is highly expressed in bovine endometrium, was reported to have PGF(2α) synthase activity, and recently, the paper entitled 'Prostaglandin F(2α) synthase activities of AKR 1B1, 1B3 and 1B7' was reported by Kabututu et al. (J. Biochem.145, 161-168, 2009). Clones that had already been registered in a database as aldose reductases (AKR1B1, 1B3, and 1B7) were expressed in Escherichia coli, and these enzymes were found to have PGF(2α) synthase activity. Moreover, in the above-cited article, the effects of inhibitors specific for aldose reductase on the PGF(2α) synthase activity of AKR1B were discussed. Here, I present an overview of various PGF/PGF(2α) synthases including those of AKR1B subfamily that have been reported until now.

Preferential localization of prostamide/prostaglandin F synthase in myelin sheaths of the central nervous system.

Prostaglandin (PG) F(2α) is a product of cyclooxygenase (COX)-catalyzed metabolism of arachidonic acid and exerts biological functions in various tissues. Prostaglandin ethanolamide (prostamide) F(2α) is a COX-2-catalyzed metabolite of arachidonoyl ethanolamide (anandamide) that induces pharmacological actions in ocular tissues. Although PGF(2α) is one of the most abundant prostaglandins in the brain, function of PGF(2α) in the central nervous system (CNS) has not been extensively investigated. Recently identified prostamide/PGF synthase catalyzes the reductions of prostamide H2 to prostamide F(2α) and PGH2 to PGF(2α), chiefly in the CNS. We examined tissue distribution of the enzyme in the CNS by immunohistochemistry, double immunofluorescence, and immuno-electron microscopy. We confirmed histological findings by immunofluorescence analyses of brain cell cultures. Prostamide/PGF synthase was expressed preferentially in the white matter bundles of the entire CNS of adult mice with less marked expression in neuronal cell bodies. The enzyme was colocalized with myelin basic protein (MBP) in myelin sheaths but not in axons. At the ultrastructural level, the enzyme was localized to myelin sheaths. Expression of the enzyme increased between P9 and P14 during the postnatal development, presumably in accordance with myelinogenesis. Cultured oligodendrocytes at 7 days in vitro expressed the enzyme in cytoplasmic processes where the enzyme was colocalized with MBP. Immunoreactivity for COX-2 was detected in white matter and cultured oligodendrocytes. Relatively selective localization of prostamide/PGF synthase suggests that myelin sheaths of the CNS may serve as the sites for producing prostamide F(2α) and/or PGF(2α), which may contribute to the formation and maintenance of central myelin.

Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily.

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.

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.