Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE₂ production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE₂ formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA₂, COX, and mPGES-1, in PGE₂ synthesis pathway, and that these enzymes contribute to the elevation of PGE₂ level when one COX isoform is absent.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

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. 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. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting 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 interleukin 1 receptor antagonist (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, whereas COX-3 and mPGES-2 mRNA expression was not modified. 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. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling,Inflammation, Adaptive Thermogenesis and Lipolysis

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE₂ levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE₂ as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE₂ significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-β. In addition, PGE₂ inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE₂ anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE₂ induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE₂ might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE₂ inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE₂ as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.     

Membrane-bound prostaglandin E synthase-1-mediated prostaglandin E2 production by osteoblast plays a critical role in lipopolysaccharide-induced bone loss associated with inflammation

PGE(2) acts as a potent stimulator of bone resorption in several disorders including osteoarthritis and periodontitis. Three PGE synthases (PGES) were isolated for PGE(2) production, but which PGES has the major role in inflammatory bone resorption is still unclear. In this study, we examined the role of PGE(2) in LPS-induced bone resorption using membrane-bound PGES (mPGES)-1-deficient mice (mPges1(-/-)). In osteoblasts from wild-type mice, PGE(2) production was greatly stimulated by LPS following the expression of cyclooxygenase 2 and mPGES-1 mRNA, whereas no PGE(2) production was found in osteoblasts from mPges1(-/-). LPS administration reduced the bone volume in wild-type femur that was associated with an increased number of osteoclasts. In mPges1(-/-), however, LPS-induced bone loss was reduced. We next examined whether mPGES-1 deficiency could alter the alveolar bone loss in LPS-induced experimental periodontitis. LPS was injected into the lower gingiva and bone mineral density of alveolar bone was measured. LPS induced the loss of alveolar bone in wild-type, but not in mPges1(-/-) mice, suggesting an mPGES-1 deficiency resistant to LPS-induced periodontal bone resorption. To understand the pathway of LPS-induced PGE(2) production in osteoblast, we used C3H/HeJ mice with mutated tlr4. Osteoblasts from C3H/HeJ mice did not respond to LPS, and PGE(2) production was not altered at all. LPS-induced bone loss in the femur was also impaired in C3H/HeJ mice. Thus, LPS binds to TLR4 on osteoblasts that directly induce mPGES-1 expression for PGE(2) synthesis, leading to subsequent bone resorption. Therefore, mPGES-1 may provide a new target for the treatment of inflammatory bone disease.     

Dienogest,a synthetic progestin,inhibits prostaglandin E2 production and aromatase expression by human endometrial epithelial cells in a spheroid culture system

Prostaglandin E₂ (PGE₂) is a major mediator in the pathophysiology, and pathogenesis of gynecological diseases associated with abnormal endometrial disease with proliferation and inflammation, such as endometriosis. In this study, we investigated the effect of dienogest, a selective progesterone receptor agonist, on PGE₂ production and the expression of aromatase, an estrogen synthase, in human immortalized endometrial epithelial cells. Compared with monolayer culture, the cells showed enhanced PGE₂ production and expression of the PGE₂ synthases cyclooxygenase-2 (COX-2), and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in a spheroid culture system. Dienogest inhibited PGE₂ production and this effect was reversed by RU486, a progesterone receptor antagonist. Dienogest inhibited the PGE₂ synthases mRNA and protein expression, and the nuclear factor-κB activation. Moreover, the suppressive effect of dienogest on PGE₂ production was sustained 24 h after the drug was withdrawn. Dienogest but not COX inhibitors inhibited aromatase expression. These results suggest that progesterone receptor activation reduces the gene expressions of COX-2, mPGES-1, and aromatase. Our findings suggest that the pharmacological mechanism of dienogest includes the direct inhibition of PGE₂ synthase and aromatase expression and may contribute to the therapeutic effect on the progression of endometriosis.     

Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H₂ (PGH₂) to PGE₂, is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE₂ production. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes

Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E₁, D₁, F_1α, E₂, D₂, or F_2α. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment.Maximum stimulation of cAMP levels was observed with PGD₂, smaller increases with PGE₂ and relatively transient rises with PGF_2α which were of low significance, but confirm earlier data. Similar results were observed with PGD₁, PGE₁, and PGF_1α with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD₂ and PGE₂. With PGF_2α, maximum cGMP levels were noted after some delay.All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD₂ treatment.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

Regional and species differences in endogenous prostaglandin biosynthesis by brain homogenates

Endogenously formed prostaglandins (PGs) D₂, E₂ and F_2α were determined in homogenates of brain regions from rat, guinea-pig, rabbit and cat, using gas-chromatography-mass spectrometry. The main PGs formed in the brain regions of the rat were PGD₂, in the guinea-pig PGD₂ and PGF_2α, in the rabbit PGF_2α and in the cat PGE₂. Brain regions from the same animal species showed the same pattern of PG formation. They varied, however, in the amount of total PGs formed, the limbic system and the cerebral cortex being highest and cerebellum lowest.     

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.     

Influences of prostaglandins on electrophoretic mobility and aggregation of rabbit platelets

Influences of prostaglandin(PG)s on electrophoretic mobilities and aggregation of rabbit platelets were studied. The PGs studied (PGI₂, PGE₁, PGD₂, PGE₂, PGF_2α, PGA₂ and PGA₁) had no effect on platelet electrophoretic mobility. However, both PGE₁ and PGI₂ in 0.3 and 3.0 μM inhibited ADP-induced aggregation and ADP-induced decrease in the mobility. PGD₂ in 0.3 and 3.0, and PGE₂ in 30 μM inhibited the aggregation but did not depress the ADP-induced decrease in the mobility. PGF_2α, PGA₂ and PGA₁ had no effect on the decrease in electrophoretic mobility and on the aggregation caused by ADP.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE₁, PGE₂ and PGI₂, but not PGD₂ or PGF_2α, increased intracellular cAMP concentrations. At maximal concentrations (10⁻⁵ tthe effects of PGE₂ plus PGI₂ (or PGE₁), but not of PGI₂ plus PGE₁, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10⁻⁵ PGE₂. PGs, when tested at concentrations (e.g. 10⁻⁹ ) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10⁻⁵ ), the effects of AVP plus PGE₁, PGE₂, PGI₂ or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

The Role of Prostaglandins and COX-Enzymes in Chondrogenic Differentiation of ATDC5 Progenitor Cells

ObjectivesNSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation.MethodsATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses.ResultsInhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD₂ and PGE₂ followed the COX-2 expression pattern, whereas PGF_2α and TXA₂ levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD₂ and PGF_2α in the COX-1 inhibited condition. Addition of PGE₂ and PGF_2α resulted in increased expression of chondrogenic markers, whereas TXA₂ increased expression of hypertrophic markers.ConclusionsOur findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production.     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.