cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis

A number of studies have identified cytosolic prostaglandin E(2) synthase (cPGES)/p23 as a cytoplasmic protein capable of metabolism of prostaglandin E(2) (PGE(2)) from the cyclooxygenase metabolite prostaglandin endoperoxide (PGH(2)). However, this protein has also been implicated in a number of other pathways, including stabilization of the glucocorticoid receptor (GR) complex. To define the importance of the functions assigned to this protein, mice lacking detectible cPGES/p23 expression were generated. cPGES/p23(-/-) pups die during the perinatal period and display retarded lung development reminiscent of the phenotype of GR-deficient neonates. Furthermore, GR-sensitive gluconeogenic enzymes are not induced in the prenatal period. However, unlike GR-deficient embryos, cPGES/p23(-/-) embryos are small and a proliferation defect is observed in cPGES/p23(-/-) fibroblasts. Analysis of arachidonic acid metabolites in embryonic tissues and primary fibroblasts failed to support a function for this protein in PGE(2) biosynthesis. Thus, while the growth retardation of the cPGES/p23(-/-) pups and decreased proliferation of primary fibroblasts identify functions for this protein in addition to GR stabilization, it is unlikely that these functions include metabolism of PGH(2) to PGE(2).     

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.     

Regulation of prostaglandin E synthases: effects of siRNA-mediated inhibition of microsomal prostaglandin E synthase-1

Prostaglandin E2 (PGE2) is a key mediator involved in several inflammatory conditions. In this study, we investigated the expression and regulation of the terminal PGE2 synthesizing enzyme prostaglandin E synthases (mPGES-1, mPGES-2 and cPGES) in gingival fibroblasts stimulated with pro-inflammatory cytokines. We used siRNA knockdown of mPGES-1 to elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production. The cytokines TNFalpha and IL-1beta increased protein expression and activity of mPGES-1, accompanied by increased COX-2 expression and PGE2 production. The isoenzymes mPGES-2 and cPGES, constitutively expressed at mRNA and protein levels, were unaffected by the pro-inflammatory cytokines. We show for the first time that treatment with mPGES-1 siRNA down-regulated the cytokine-induced mPGES-1 protein expression and activity. Interestingly, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas PGF(2alpha) levels were enhanced. Neither mPGES-2 nor cPGES expression was affected by siRNA silencing of mPGES-1. Dexamethasone and MK-886 both inhibited the cytokine-induced mPGES-1 expression while mPGES-2 and cPGES expression remained unaffected. In conclusion, mPGES-1 siRNA down-regulates mPGES-1 expression, and neither mPGES-2 nor cPGES substituted for mPGES-1 in a knockdown setting in gingival fibroblasts. Moreover, mPGES-1 siRNA did not affect PGE2 levels, whereas PGF(2alpha) increased, suggesting a compensatory pathway of PGE2 synthesis when mPGES-1 is knocked down.     

Regulation of cytosolic prostaglandin E2 synthase by 90-kDa heat shock protein

Cytosolic prostaglandin (PG) E(2) synthase (cPGES) is constitutively expressed in a wide variety of cells and converts cyclooxygenase (COX)-1-derived PGH(2) to PGE(2). Given the fact that cPGES is identical to p23, a heat shock protein 90 (Hsp90)-binding protein, we herein examined the effect of Hsp90 on PGE(2) generation by cPGES. Incubation of cPGES with Hsp90 resulted in a significant increase in PGES activity in vitro. Association of cPGES with Hsp90 was increased in cells stimulated with A23187 or bradykinin, accompanied by concomitant increases in cPGES activity and PGE(2) production. Moreover, treatment of cells with Hsp90 inhibitors, which destabilized the cPGES/Hsp90 complex, reduced cPGES activity and PGE(2) production to basal levels. These results suggest that the regulation of cPGES activity in cells depends on its association with Hsp90 and provide the first line of evidence that eicosanoid biosynthesis is under the control of the molecular chaperone.     

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.     

Cytoplasmic prostaglandin E2 synthase is dominantly expressed in cultured KAT-50 thyrocytes,cells that express constitutive prostaglandin-endoperoxide H synthase-2. Basis for low protaglandin E2 production

The recent identification and cloning of two glutathione-dependent prostaglandin E(2) synthase (PGES) genes has yielded important insights into the terminal step of PGE(2) synthesis. These enzymes form efficient functional pairs with specific members of the prostaglandin-endoperoxide H synthase (PGHS) family. Microsomal PGES (mPGES) is inducible and works more efficiently with PGHS-2, the inflammatory cyclooxygenase, while the cytoplasmic isoform (cPGES) pairs functionally with PGHS-1, the cyclooxygenase that ordinarily exhibits constitutive expression. KAT-50, a well differentiated thyroid epithelial cell line, expresses high levels of PGHS-2 but surprisingly low levels of PGE(2) when compared with human orbital fibroblasts. Moreover, PGHS-1 protein cannot be detected in KAT-50. We report here that KAT-50 cells express high basal levels of cPGES but mPGES mRNA and protein are undetectable. Thus, KAT-50 cells express the inefficient PGHS-2/cPGES pair, and this results in modest PGE(2) production. The high levels of cPGES and the absence of mPGES expression result from dramatic differences in the activities of their respective gene promoters. When mPGES is expressed in KAT-50 by transiently transfecting the cells, PGE(2) production is up-regulated substantially. These observations indicate that naturally occurring cells can express a suboptimal profile of PGHS and PGES isoforms, resulting in diminished levels of PGE(2) generation.     

Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Up-regulation of prostaglandin E2 synthesis by interleukin-1beta in human orbital fibroblasts involves coordinate induction of prostaglandin-endoperoxide H synthase-2 and glutathione-dependent prostaglandin E2 synthase expression

Prostaglandin E(2) (PGE(2)) production involves the activity of a multistep biosynthetic pathway. The terminal components of this cascade, two PGE(2) synthases (PGES), have very recently been identified as glutathione-dependent proteins. cPGES is cytoplasmic, apparently identical to the hsp90 chaperone, p23, and associates functionally with prostaglandin-endoperoxide H synthase-1 (PGHS-1), the constitutive cyclooxygenase. A second synthase, designated mPGES, is microsomal and can be regulated. Here we demonstrate that mPGES and PGHS-2 are expressed at very low levels in untreated human orbital fibroblasts. Interleukin (IL)-1beta treatment elicits high levels of PGHS-2 and mPGES expression. The induction of both enzymes occurs at the pretranslational level, is the consequence of enhanced gene promoter activities, and can be blocked by dexamethasone (10 nm). SC58125, a PGHS-2-selective inhibitor, could attenuate the induction of mPGES, suggesting a dependence of this enzyme on PGHS-2 activity. IL-1beta treatment activates p38 and ERK mitogen-activated protein kinases. Induction of both mPGES and PGHS-2 was susceptible to either chemical inhibition or molecular interruption of these pathways with dominant negative constructs. These results indicate that the induction of PGHS-2 and mPGES by IL-1beta underlies robust PGE(2) production in orbital fibroblasts.     

Knockout mice lacking cPGES/p23, a constitutively expressed PGE2 synthetic enzyme, are peri-natally lethal

Cytosolic prostaglandin (PG) E synthase (cPGES) is constitutively expressed in various cells and regulates cyclooxygenase (COX)-1-dependent immediate PGE(2) generation. Its primary structure is identical to co-chaperone p23, a heat shock protein 90 (Hsp90)-binding protein. We have revealed that Hsp90 regulated both cPGES/p23 and its client protein kinase CK2. In this study, in order to examine the role of cPGES/p23 in vivo, we generated mice deficient in cPGES/p23 by a targeted disruption of exons 2 and 3, containing Tyr9, which is essential for catalytic activity. Heterozygotes are viable, fertile, and appear normal, despite a decrease in cPGES/p23 protein level. A generation of offsprings derived from intercrosses of cPGES/p23 homozygous mice revealed that 109, 247, and 10 pups were wild type, heterozygous, and homozygous, respectively; however, all homozygotes died at birth. The absence of viable null mutants, with heterozygotes and wild-type offspring obtained at a ratio of approximately 2:1, indicated that homozygosity for the cPGES/p23 null mutant leads to peri-natal lethality. Embryos homozygous for cPGES/p23-null had lower body weights than wild-type embryos, and abnormal morphology of skin and lungs. Moreover, the PGE(2) content in the lungs of cPGES/p23-null embryos was lower than that of the wild type. These results indicate that cPGES-derived PGES is involved in the normal development of mouse embryonic lung.     

Inhibition of Rho modulates cytokine-induced prostaglandin E2 formation in renal mesangial cells

Stimulation of rat mesangial cells for 24 h with interleukin-1beta (IL- 1beta) plus forskolin (Fk) leads to a marked increase in prostaglandin E2 (PGE2) synthesis. This effect is further enhanced by the small G-protein Rho inhibitor toxin A. A similar increase in PGE2 formation is obtained with Y27632, a Rho-dependent kinase inhibitor, and with lovastatin, a hydroxymethylglutaryl-coenzyme A inhibitor which depletes cells from geranylgeranyl moieties and thus blocks Rho activation. In parallel to the increased PGE2 synthesis, a potentiation of IL-1beta-induced secretory group IIA phospholipases A2 (sPLA2-IIA) protein expression also occurs by Rho inhibition. However, only toxin A triggers an increased sPLA2-IIA activity consistent with the elevated levels of protein expression, whereas Y27632 and lovastatin rather reduced IL-1beta-induced sPLA2-IIA activity. In vitro activity studies reveal that Y27632 and lovastatin can directly block sPLA2-IIA enzyme activity in a concentration-dependent manner. Interestingly, in the absence of IL-1beta/Fk stimulation and the lack of sPLA2-IIA protein expression, all Rho inhibitors exert a small but significant increase in PGE2 formation suggesting that additional PLA2s or downstream enzymes like cyclooxygenases or prostaglandin synthases may be activated by Rho inhibitors. Western blot analyses of toxin A-, Y27632- and lovastatin-stimulated cells reveal that the cytosolic group IV PLA2 (cPLA2) and the cytosolic PGE2 synthase (cPGES), but not the sPLA2-IIA, cyclooxygenase-2 or the microsomal PGE2 synthase (mPGES), are upregulated compared to unstimulated cells. Furthermore, the Rho inhibitors induced arachidonic acid release from intact cells which is blocked by the cPLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP). In summary, these data show that inhibition of the small G-protein Rho, either by toxin A, lovastatin, or Y27632, exert a dual effect on mesangial cells: (i) in the absence of an inflammatory stimulus it activates the constitutive cPLA2 and cPGE2 synthase and generates low amount of PGE2. (ii) In the presence of inflammatory cytokines it potentiates sPLA2-IIA expression and subsequent PGE2 formation. In addition, we identified lovastatin and Y27632 as direct inhibitors of sPLA2-IIA in a cell-free system.     

Expression and regulation of cytosolic prostaglandin E synthase in mouse uterus during the peri-implantation period

Prostaglandin E(2) (PGE(2)) is considered important for blastocyst spacing, implantation, and decidualization in rodent uteri. PGE synthase (PGES) catalyzes the isomerization of PGH(2) to PGE(2). Two isoforms of PGES exist: microsomal PGES (mPGES) and cytosolic PGES (cPGES); however, the expression and regulation of cPGES in the mammalian uterus during early pregnancy are still unknown. The aim of this study was to investigate the differential expression of cPGES in mouse uterus during early pregnancy and its regulation under different conditions using in situ hybridization and immunohistochemistry. A strong level of cPGES mRNA signal was exhibited in the stromal cells at the implantation site on Day 5 of pregnancy, whereas cPGES immunostaining was strongly detected in the luminal epithelium. The signals for both cPGES mRNA and immunostaining were strongly detected in the decidualized cells from Days 6-8 of pregnancy. A basal level of cPGES mRNA signal and immunostaining was exhibited in the uterus in delayed implantation. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, cPGES mRNA signal was strongly detected in the stroma underlying the luminal epithelium at the implantation site, and cPGES immunostaining was strongly observed in the luminal epithelium surrounding the implanting blastocyst. A strong cPGES mRNA signal and immunostaining were detected in decidualized cells under artificial decidualization, whereas only a basal level of cPGES mRNA signal and immunostaining were observed in the control horn. Our data suggest that cPGES may play an important role during implantation and decidualization.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

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. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Intracellular-specific colocalization of prostaglandin E2 synthases and cyclooxygenases in the brain

Prostaglandin E2 (PGE2) is the major primary prostaglandin generated by brain cells. However, the coordination and intracellular localization of the cyclooxygenases (COXs) and prostaglandin E synthases (PGESs) that convert arachidonic acid to PGE2 in brain tissue are not known. We aimed to determine whether microsomal and cytosolic PGES (mPGES-1 and cPGES) colocalize and coordinate activity with either COX-1 or COX-2 in brain tissue, particularly during development. Importantly, we found that cytosolic PGES also associates with microsomes (cPGES-m) from the cerebrum and cerebral vasculature of the pig and rat as well as microsomes from various cell lines; this seemed dependent on the carboxyl terminal 35-amino acid domain and a cysteine residue (C58) of cPGES. In microsomal membranes from the postnatal brain and cerebral microvessels of mature animals, cPGES-m colocalized with both COX-1 and COX-2, whereas mPGES-1 was undetectable in these microsomes. Accordingly, in this cell compartment, cPGES could coordinate its activity with COX-2 and COX-1 (partly inhibited by NS398); albeit in microsomes of the brain microvasculature from newborns, mPGES-1 was also present. In contrast, in nuclei of brain parenchymal and endothelial cells, mPGES-1 and cPGES colocalized exclusively with COX-2 (determined by immunoblotting and immunohistochemistry); these PGESs contributed to conversion of PGH2 into PGE2. Hence, contrary to a previously proposed model of exclusive COX-2/mPGES-1 coordination, COX-2 can coordinate with mPGES-1 and/or cPGES in the brain, depending on the cell compartment and the age group.     

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.     

Sex hormones induce a gender-related difference in renal expression of a novel prostaglandin transporter, OAT-PG, influencing basal PGE2 concentration

Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE(2) and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE(2) clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE(2), and 15-PGDH in the renal cortex. Tissue PGE(2) concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE(2) concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE(2) concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE(2) clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE(2) clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE(2) in the kidney.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.