Genetic variation in prostaglandin E2 synthesis and signaling, prostaglandin dehydrogenase, and the risk of colorectal adenoma.
ABSTRACT: Prostaglandins are important inflammatory mediators; prostaglandin E2 (PGE2) is the predominant prostaglandin in colorectal neoplasia and affects colorectal carcinogenesis. Prostaglandins are metabolites of omega-6 and omega-3 polyunsaturated fatty acids; their biosynthesis is the primary target of nonsteroidal anti-inflammatory drugs (NSAID), which reduce colorectal neoplasia risk.We investigated candidate and tagSNPs in PGE2 synthase (PGES), PGE2 receptors (EP2 and EP4), and prostaglandin dehydrogenase (PGDH) in a case-control study of adenomas (n = 483) versus polyp-free controls (n = 582) and examined interactions with NSAID use or fish intake, a source of omega-3 fatty acids.A 30% adenoma risk reduction was observed for EP2 4950G>A (intron 1; OR(GA/AA vs. GG), 0.71; 95% confidence interval, 0.52-0.99). For the candidate polymorphism EP4 Val294Ile, increasing fish intake was associated with increased adenoma risk among those with variant genotypes, but not among those with the Val/Val genotype (P(interaction) = 0.02). An interaction with fish intake was also observed for PGES -664A>T (5' untranslated region; P(interaction) = 0.01). Decreased risk with increasing fish intake was only seen among those with the AT or TT genotypes (OR(>2 t/wk vs. <1 t/wk), 0.56; 95% confidence interval, 0.28-1.13). We also detected interactions between NSAIDs and EP2 9814C>A (intron 1) and PGDH 343C>A (intron 1). However, none of the observed associations was statistically significant after adjustment for multiple testing. We investigated potential gene-gene interactions using the Chatterjee 1 degree of freedom Tukey test and logic regression; neither method detected significant interactions.These data provide little support for associations between adenoma risk and genetic variability related to PGE(2), yet suggest gene-environment interactions with anti-inflammatory exposures.
Project description: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.
Project description:Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Previously, we reported that in macrophages (M?s), infection with avirulent Mtb H37Ra resulted in inhibition of necrosis by an inhibitory effect on mitochondrial permeability transition via the PGE2 receptor EP2. However, human M?s also express EP4, a PGE2 receptor functionally closely related to EP2 that also couples to stimulatory guanine nucleotide binding protein, but the functional differences between EP2 and EP4 in Mtb-infected M?s have been unclear. EP4 antagonist addition to H37Ra-infected M?s inhibited the expression of cyclooxygenase 2 (COX2) and microsomal prostaglandin E synthase-1 (mPGES-1), which are involved in PGE2 production. Moreover, H37Ra infection induced PGE2 production through the Toll-like receptor (TLR) 2/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Induction of COX2 and mPGES-1 expression by TLR2 stimulation or Mtb infection was increased after additional stimulation with EP4 agonist. Hence, in Mtb-infected M?s, PGE2 production induced by pathogen recognition receptors/p38 MAPK signaling is up-regulated by EP4-triggered signaling to maintain an effective PGE2 concentration.
Project description:The cervix represents a formidable structural barrier for successful induction of labor. Approximately 10% of pregnancies undergo induction of cervical ripening and labor with prostaglandin (PG) E2 or PGE analogs, often requiring many hours of hospitalization and monitoring. On the other, preterm cervical ripening in the second trimester predicts preterm birth. The regulatory mechanisms of this paradoxical function of the cervix are unknown. Here, we show that PGE2 utilizes cell-specific EP2 receptor-mediated increases in Ca2+ to dephosphorylate and translocate HDAC4 to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH). The crucial role of 15-PGDH in cervical ripening was confirmed in vivo. Although PGE2 or PGDH inhibitor alone did not alter gestational length, treatment with PGDH inhibitor+PGE2 or metabolism-resistant dimethyl-PGE2 resulted in preterm cervical ripening and delivery in mice. The ability of PGE2 to selectively auto-amplify its own concentrations in stromal cells by signaling transcriptional repression of 15-PGDH elucidates long-sought-after molecular mechanisms that govern prostaglandin action in the cervix. This is the first report detailing unique mechanisms of action in the cervix and serves as a catalyst for (i) the use of PGDH inhibitors to initiate, or amplify low-dose PGE2-mediated cervical ripening, or (ii) EP2 receptor antagonists, HDAC4 inhibitors, and 15-PGDH activators to prevent preterm cervical ripening and preterm birth. Overall design: human cervical stromal cells were treated with DMSO (vehicle) or PGE2 (100 nM) for 1 h or 24 h followed by RNA sequencing
Project description:Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS.Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS.PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect.These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.
Project description:BACKGROUND: The regulatory mechanisms of the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2) in human articular chondrocytes have not been clarified. We investigated the effect of prostaglandin E2 (PGE2) on CTGF/CCN2 expression in chondrocytes. FINDINGS: Articular cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were isolated and cultured in vitro. Chondrocytes were stimulated with PGE2, PGE receptor (EP)-specific agonists, or interleukin (IL)-1. CTGF expression was analyzed using quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The inhibitory effects of EP receptor antagonists (for EP2 and EP4) against PGE2 stimulation were also investigated. Stimulation of chondrocytes with PGE2 or IL-1 significantly suppressed CTGF expression. The suppressive effect of PGE2 was reproduced by EP2/EP4 receptor agonists but not by EP1/EP3 receptor agonists, and was partially blocked by an EP4 receptor antagonist, suggesting that the EP4 receptor has a dominant role. CONCLUSIONS: PGE2 may be involved in the regulation of CTGF/CCN2 expression in human articular chondrocytes via the EP4 receptor. Elucidation of EP4-mediated signaling in chondrocytes may contribute to a better understanding of the effects of PGE2 in arthritis.
Project description:Cyclooxygenase-2 (COX-2) triggers pro-inflammatory processes that can aggravate neuronal degeneration and functional impairments in many neurological conditions, mainly via producing prostaglandin E2 (PGE2) that activates four membrane receptors, EP1-EP4. However, which EP receptor is the culprit of COX-2/PGE2-mediated neuronal inflammation and degeneration remains largely unclear and presumably depends on the insult types and responding components. Herein, we demonstrated that COX-2 was induced and showed nuclear translocation in two neuronal cell lines - mouse Neuro-2a and human SH-SY5Y - after treatment with neurotoxin 6-hydroxydopamine (6-OHDA), leading to the biosynthesis of PGE2 and upregulation of pro-inflammatory cytokine interleukin-1?. Inhibiting COX-2 or microsomal prostaglandin E synthase-1 suppressed the 6-OHDA-triggered PGE2 production in these cells. Treatment with PGE2 or EP2 selective agonist butaprost, but not EP4 agonist CAY10598, increased cAMP response in both cell lines. PGE2-initiated cAMP production in these cells was blocked by our recently developed novel selective EP2 antagonists - TG4-155 and TG6-10-1, but not by EP4 selective antagonist GW627368X. The 6-OHDA-promoted cytotoxicity was largely blocked by TG4-155, TG6-10-1 or COX-2 selective inhibitor celecoxib, but not by GW627368X. Our results suggest that PGE2 receptor EP2 is a key mediator of COX-2 activity-initiated cAMP signaling in Neuro-2a and SH-SY5Y cells following 6-OHDA treatment, and contributes to oxidopamine-mediated neurotoxicity.
Project description:Incidence of kidney cancer is on the rise, and a better understanding of molecular mechanisms involved in the cancer invasion and metastasis is required for the development of curative therapeutics. In this study, we report that the proinflammatory cytokine prostaglandin E2 (PGE2) induces the malignant SN12C, but not benign HK2 kidney cell invasion. The PGE2 increases SN12C cell invasion through a signal pathway that encompasses EP2 and EP4, Akt, small GTPase RalA and Ral·GTP inactivator RGC2. The results support the idea that targeted interference of EP2/EP4 signal to RalA·GTP may provide benefit to patients diagnosed with advanced kidney cancer.
Project description:UNLABELLED:Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE2) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE2 is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets. SIGNIFICANCE STATEMENT:Brain function critically depends on a permanent spatiotemporal match between neuronal activity and blood supply, known as NVC. In the cerebral cortex, prostaglandins are major contributors to NVC. However, their biochemical identity remains elusive and their cellular origins are still under debate. Although astrocytes can induce vasodilations through the release of prostaglandins, the recruitment of this pathway during sensory stimulation is questioned. Using multidisciplinary approaches from single-cell reverse transcriptase-PCR, mass spectrometry, to ex vivo and in vivo pharmacology and optogenetics, we provide compelling evidence identifying PgE2 as the main prostaglandin in NVC, pyramidal neurons as their main cellular source and the vasodilatory EP2 and EP4 receptors as their main targets. These original findings will certainly change the current view of NVC.
Project description:We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-? (TGF-?) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-? in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-?-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-?-stimulated expression of collagen A1, fibronectin, and ?-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-?. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.
Project description:In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2? occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog.Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2?-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion.Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments.The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion.