mPGES-1 Deficiency Research Articles

Obesity-associated renal alterations are mediated by Microsomal Prostaglandin E Synthase-1 (mPGES-1) in a sex-dependent manner.

Introduction: Obesity is characterized by a chronic-low grade inflammation that causes pathophysiological alterations in different target organs, contributing, among others, to the development of renal damage. Microsomal Prostaglandin E Synthase 1 (mPGES-1) is an inducible isomerase responsible for the overexpression of prostaglandin E2 (PGE2) under inflammatory conditions, including obesity. Inhibition of PGE2 production by non-steroidal anti-inflammatory drugs, that block cyclooxygenase-2 (COX-2) cascade, limits inflammatory processes. However, it can be associated with side- effects as nephrotoxicity due to the inhibition of other prostaglandins production. New drugs targeting mPGES-1 have shown promising results in different experimental models. Nevertheless, to our knowledge, efficacy of inhibition downstream COXs in the renal impact of obesity remains unexplored. Objective: To elucidate whether mPGES-1 depletion could prevent the development of renal alterations in an experimental model of obesity. Methods: We have developed a model of HFD-induced obesity (60% fat, 13 weeks) in males and females mPGES-1-/- and their control littermates mPGES-1+/+ mice. Renal structural alterations and changes in mRNA expression were studied by histological analysis and q-RT-PCR, respectively. Results: IHFD decreased renal mPGES-1 gene expression in females but not in males. On the contrary, mPGES-1 expression was upregulated in perirenal adipose tissue (PRAT) of both sexes. In females, HFD induced kidney and PRAT hypertrophy that was prevented by the absence of mPGES-1; whereas in males, HFD did not modify kidney weight, while increasing PRAT weight significantly more in mPGES-1+/+ than in mPGES-1-/- mice. Rt-qPCR studies revealed that HFD induced renal damage, inflammation and fibrosis in mPGES-1+/+ male mice, which was not observed in mPGES-1 deficient mice. In agreement, histological studies showed that HFD induced glomerular hypertrophy and renal fibrosis only in mPGES-1+/+ HFD males. These alterations were not likely mediated by oxidative stress. Of note, females were protected against renal inflammatory and fibrotic alterations induced by HFD. Additionally, mPGES-1 deficiency prevented HFD- induced PRAT inflammation, fibrosis and hypoxia in both males and females. HFD did not modify serum proteinuria in any sex. Conclusion: mPGES-1 participates in the renal alterations associated with obesity in a s ex-dependent manner.

Facilitation of colonic T cell immune responses is associated with an exacerbation of dextran sodium sulfate\u2013induced colitis in mice lacking microsomal prostaglandin E synthase-1

BackgroundMicrosomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that acts downstream of cyclooxygenase and plays a major role in inflammation by converting prostaglandin (PG) H2 to PGE2. The present study investigated the effect of genetic deletion of mPGES-1 on the development of immunologic responses to experimental colitis induced by dextran sodium sulfate (DSS), a well-established model of inflammatory bowel disease (IBD).MethodsColitis was induced in mice lacking mPGES-1 (mPGES-1−/− mice) and wild-type (WT) mice by administering DSS for 7 days. Colitis was assessed by body weight loss, diarrhea, fecal bleeding, and histological features. The colonic expression of mPGES-1 was determined by real-time PCR, western blotting, and immunohistochemistry. The impact of mPGES-1 deficiency on T cell immunity was determined by flow cytometry and T cell depletion in vivo.ResultsAfter administration of DSS, mPGES-1−/− mice exhibited more severe weight loss, diarrhea, and fecal bleeding than WT mice. Histological analysis further showed significant exacerbation of colonic inflammation in mPGES-1−/− mice. In WT mice, the colonic expression of mPGES-1 was highly induced on both mRNA and protein levels and colonic PGE2 increased significantly after DSS administration. Additionally, mPGES-1 protein was localized in the colonic mucosal epithelium and infiltrated inflammatory cells in underlying connective tissues and the lamina propria. The abnormalities consistent with colitis in mPGES-1−/− mice were associated with higher expression of colonic T-helper (Th)17 and Th1 cytokines, including interleukin 17A and interferon-γ. Furthermore, lack of mPGES-1 increased the numbers of Th17 and Th1 cells in the lamina propria mononuclear cells within the colon, even though the number of suppressive regulatory T cells also increased. CD4+ T cell depletion effectively reduced symptoms of colitis as well as colonic expression of Th17 and Th1 cytokines in mPGES-1−/− mice, suggesting the requirement of CD4+ T cells in the exacerbation of DSS-induced colitis under mPGES-1 deficiency.ConclusionsThese results demonstrate that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis by affecting the development of colonic T cell–mediated immunity. mPGES-1 might therefore impact both the intestinal inflammation and T cell–mediated immunity associated with IBD.

Gene Deletion of Microsomal Prostaglandin E Synthase-1 Suppresses Chemically Induced Skin Carcinogenesis.

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) is a terminal enzyme in PGE2 synthesis and highly expressed in several cancers. In this study, to reveal the involvement of mPGES-1 in skin carcinogenesis, the effect of mPGES-1 deficiency on two-stage skin carcinogenesis in mice was investigated. A two-stage skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter was applied on mPGES-1 knockout (KO) mice and littermate wild-type mice of a Balb/c genetic background. DMBA/TPA-induced skin carcinogenesis was suppressed in mPGES-1 KO mice. The induction of IL-17 and other inflammatory cytokines by TPA was also suppressed by mPGES-1 deficiency, although DMBA-induced apoptosis was not affected. mPGES-1 promotes chemically induced skin carcinogenesis and might play an important role in the TPA-induced promotion phase of the two-stage skin carcinogenesis model. mPGES-1 inhibition may be a therapeutic target for skin cancer.

Involvement of prostacyclin synthase in high-fat-diet-induced obesity

Prostacyclin (PGI2) synthase (PGIS) functions downstream of inducible cyclooxygenase COX-2 in the PGI2 biosynthetic pathway. Although COX-2 and PGI2 receptor (IP) are known to be involved in adipogenesis and obesity, the involvement of PGIS has not been fully elucidated. In this study, we examined the role of PGIS in adiposity by using PGIS-deficient mice. Although PGIS deficiency did not affect in vitro adipocyte differentiation, when fed a high-fat diet (HFD), PGIS knockout (KO) mice showed reductions in both body weight gain and epididymal fat mass relative to wild-type (WT) mice. PGIS deficiency might reduce HFD-induced obesity by suppressing PGI2 production. We further found that additional gene deletion of microsomal prostaglandin (PG) E synthase-1 (mPGES-1), one of the other PG terminal synthases that also functions downstream of COX-2, emphasized the metabolic phenotypes of PGIS-deficient mice. More marked reduction in obesity and improved insulin resistance were observed in PGIS/mPGES-1 double KO (DKO) mice. Since an additive increase in PGF2α level in epididymal fat was observed in DKO mice, mPGES-1 deficiency might affect adiposity by enhancing the production of PGF2α. Our immunohistochemical analysis further revealed that in adipose tissues, PGIS was expressed in vascular and stromal cells but not in adipocytes. These results suggested that PGI2 produced from PGIS-expressed stromal tissues might enhance HFD-induced obesity by acting on IP expressed in adipocytes. The balance of expressions of PG terminal synthases and the subsequent production of prostanoids might be critical for adiposity.

Abstract 5073: mPGES-1 deficiency impairs self-renewal properties of colon cancer stem cells

Abstract Dysregulation of prostaglandin E2 (PGE2) signaling is a hallmark of many cancers, including colorectal cancer (CRC). Direct suppression of inducible PGE2 synthesis by genetic deletion of Ptges1 (mPGES-1) affords dramatic cancer protection to the colon. However, the precise mechanisms by which this effect occurs remain incompletely understood. Recent evidence points to a fundamental role of PGE2 signaling in the expansion of cancer stem cells. In the present study, using an organoid system established from Apc mutant mice, we have evaluated the influence of PGE2 and related prostanoids on the viability of cancer stem cells in the colon. To establish cancer organoids, colon tumors were harvested from 16 week-old ApcΔ14/+ mice with or without mPGES-1 (D14:WT and D14:KO). Colon tumor multiplicity in D14:KO mice is approximately half the number of tumors in D14:WT mice. Tumor tissues were digested with collagenase and dispase, and cultured in Matrigel with complete organoid media. Organoid growth was scored daily over a one-week time period, and passaged. Tumor organoids generated from D14:WT and D14:KO mice showed no obvious differences in their growth characteristics nor morphological features during the first week of ex-vivo growth. After the first passage, however, the growth rate of D14:KO organoids was significantly reduced (175% vs. 80%, WT and KO, respectively), and by the second passage, D14:KO organoids failed to maintain cell viability. Immunohistochemical analysis of the organoids showed strong nuclear localization of ß-catenin and loss of Apc expression regardless of mPGES-1 genotype, indicating intact Wnt-driven cancer growth mechanisms that were independent of inducible PGE2 signaling. These observations indicate that alternative mechanisms are contributing to the compromised stem cell expansion observed in the D14:KO organoids. Since mPGES-1 is expressed within the tumor stroma, we postulated that the inactivation of mPGES-1 would cause a marked microenvironmental change. To test this possibility, we used GC-MS/MS to measure a panel of prostanoids within the tumor tissue. Targeted lipidomic analysis showed significant metabolite redirection of tissue prostanoids, with a significant increase (2-fold) in PGD2in the D14:KO mice. Interestingly, administration of PGD2 was recently shown to reduce the growth and regeneration potential of stem cells within a hair follicle organ culture system. Given new evidence that PGD2 may have a direct influence on ‘stem cell-ness' in other organ systems, we propose that the colon tumor microenvironment, deficient in inducible PGE2, may undergo permanent molecular changes that directly influence the growth characteristics and viability of cancer stem cells as reflected in our organoid culture system. New data is presented to define the long-lasting influence of eicosanoid metabolic changes on cancer-derived stem cell viability and proliferative capacity. Citation Format: Masako Nakanishi, Daniel W. Rosenberg. mPGES-1 deficiency impairs self-renewal properties of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5073.

Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mouse spleen.

The cholinergic anti-inflammatory pathway (CAP) is an innate neural reflex where parasympathetic and sympathetic nerves work jointly to control inflammation. Activation of CAP by vagus nerve stimulation (VNS) has paved way for novel therapeutic strategies in treating inflammatory diseases. Recently, we discovered that VNS mediated splenic acetylcholine (ACh) release and subsequent immunosuppression in response to LPS associated inflammation is impaired in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1) expression, a key enzyme responsible for prostaglandin E2 synthesis. Here, we have further investigated the consequences of mPGES-1 deficiency on various molecular/cellular events in the spleen which is critical for the optimal functioning of VNS in endotoxaemic mice. First, VNS induced splenic norepinephrine (NE) release in both mPGES-1 (+/+) and (-/-) mice. Compared to mPGES-1 (+/+), immunomodulatory effects of NE on cytokines were strongly compromised in mPGES-1 (-/-) splenocytes. Interestingly, while LPS increased choline acetyltransferase (ChAT) protein level in mPGES-1 (+/+) splenocytes, it failed to exert similar effects in mPGES-1 (-/-) splenocytes despite unaltered β2 AR protein expression. In addition, nicotine inhibited TNFα release by LPS activated mPGES-1 (+/+) splenocytes in vitro. However, such immunosuppressive effects of nicotine were reversed both in mPGES-1 (-/-) mouse splenocytes and human PBMC treated with mPGES-1 inhibitor. In summary, our data implicate PGE2 as an important mediator of ACh synthesis and noradrenergic/cholinergic molecular events in the spleen that constitute a crucial part of the CAP immune regulation. Our results suggest a possible link between cholinergic and PG system of CAP that may be of clinical significance in VNS treatment.

PGE2 controls Th17 and regulatory T cell function during intestinal immune responses

Abstract Eicosanoids, particularly prostaglandin E2 (PGE2), are critical molecules in the initiation and resolution of inflammation during immune responses. PGE2 is associated with modulating autoimmunity through altering the IL-23/IL-17 axis and regulatory T cells, whose balance is critical for controlling pathogenic T cells. The inducible microsomal prostaglandin E synthase 1 (mPGES1) is a membrane enzyme that regulates local PGE2 levels and is highly expressed at sites of inflammation. Hematopoietic and stromal cells express mPGES1, but its role in T cell function and intestinal biology remains underappreciated. Th17 polarized cells produce 10-fold more PGE2 than Th1 polarized cells, and this PGE2 production is mPGES1-dependent. Interestingly, high concentrations of PGE2 can shift the production from IL-17A to IFNg during Th17 polarization, and PGE2 promotes more cell proliferation and cytokine production in Th1 than in Th17 cells. Sensing of PGE2 at physiological concentrations through its EP4 receptor proved to be critical to inhibit the generation of iTregs but not Th1 or Th17 cells. In the colon, we observed that different inflammatory insults induce different PGE2 production levels, with concomitant modulation of T cell responses in the mesenteric lymph nodes. Furthermore, mPGES1 displayed opposite effects on pathogenic and regulatory T cells in the T-cell transfer colitis model, with mPGES1 deficiency limiting intestinal damage driven by effector T cells, but enhancing de novo generation of Tregs and increasing colonic lamina propria homing of CD4+FoxP3+ cells. Taken together, these data demonstrate the pleiotropic and context-dependent actions of mPGES1 and PGE2 in intestinal inflammation and immunity.

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

Paradoxical Increase in Mortality and Rupture of Intracranial Aneurysms in Microsomal Prostaglandin E2 Synthase Type 1-Deficient Mice: Attenuation by Aspirin.

Inflammation plays an important role in formation and rupture of intracranial aneurysms. Expression of microsomal prostaglandin E2 (PGE2) synthase type 1 (mPGES-1) is increased in the wall of intracranial aneurysms in humans. PGE2, a by-product of mPGES-1, is associated with inflammation and cerebrovascular dysfunction. To test the hypothesis that deletion of mPGES-1 decreases the formation and rupture of intracranial aneurysms in a murine model. Intracranial aneurysms were induced in wild-type and mPGES-1 knockout (mPGES-1 KO) mice by using a combination of deoxycorticosterone acetate-salt-induced hypertension and intracranial injection of elastase in the basal cistern. Prevalence of aneurysms, subarachnoid hemorrhage, and mortality were assessed. We also tested the effects of administration of aspirin (6 mg/kg/d) by gavage and PGE2 (1 mg/kg/d) by subcutaneous infusion. Systolic blood pressure and prevalence of aneurysm were similar in wild-type and mPGES-1 KO mice. However, mortality and the prevalence of subarachnoid hemorrhage were markedly increased in mPGES-1 KO mice (P < .05). Bone marrow reconstitution studies suggest that mPGES-1 derived from leukocytes does not appear to increase rupture of intracranial aneurysms. Aspirin, but not PGE2, attenuated the increased mortality in mPGES-1 KO mice (P < .05). Vascular mPGES-1 plays a protective role in blood vessels and attenuates rupture of cerebral aneurysms. In contrast to effects on abdominal aneurysms, mPGES-1 deficiency is associated with an increase in rupture of cerebral aneurysms and mortality, which are attenuated by low-dose aspirin.

Regulatory T cell differentiation and Th17 T cell function are regulated by mPGES-1-dependent PGE2 (IRC11P.424)

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that regulates inflammatory and immune responses. PGE2 has pleiotropic effects on many cell types, but it is still unclear how it affects T cell differentiation and function. The mPGES1 enzyme is part of the PGE2 biosynthetic pathway and, due to its inducible nature, it controls PGE2 concentrations during activation of the inflammatory program. Absence of mPGES1 causes shunting to alternate prostaglandins and changes the characteristics of the inflammatory response. We identified that, compared to WT, naïve T cells from mice with genetic deletion of mPGES1 express a 4-fold lower level of TFGbR1 and 8-fold lower level of the EP4 PGE receptor. Since TGF-b is a key cytokine for the generation of both regulatory and Th17 cells we addressed the effect of mPGES1 deficiency during in vitro T cell polarization. We observed that mPGES1-/- naïve CD4+ T cells generate less iTreg cells, while Th17 commitment is unaffected. However, when stimulating T cells with anti-CD3/CD28 from mice following immunization with type II collagen (CII), mPGES1-/- CD4+ T cells were unable to secrete IL-17A compared to its WT T cell counterparts. Furthermore, in-vivo antigen-specific Th17 responses from CII immunized mice were also impaired, showing a decrease in IL17A production in mPGES1-/- T cells compared to WT. Hence, endogenous production and sensing of PGE2 modulates T cell differentiation possibly by altering TGF-b responses.

Non-cell autonomous effects of targeting inducible PGE2 synthesis during inflammation-associated colon carcinogenesis.

Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc (Δ14/+) in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early cryptal structures.

Abstract 170: Aspirin Reduces Mortality Caused by Intracranial Aneurysm Rupture in mPGES-1 Deficient Mice

Inflammation plays an important role in formation and rupture of intracranial aneurysms. Prostaglandin (PG) E2, a by-product of microsomal prostaglandin E2 synthase (mPGES)-1, is associated with inflammation and cerebrovascular dysfunction. We hypothesized that mPGES-1 deficiency protects against rupture of intracranial aneurysms. Intracranial aneurysms were induced in mPGES-1 knockout (KO) and wild-type (WT) mice with DOCA-salt hypertension and an intracranial injection of elastase. Systolic blood pressure and water intake (1% saline) was similar in WT and mPGES-1 KO mice. All WT mice survived 3 weeks after surgery and did not develop signs of subarachnoid hemorrhage (SAH). The majority of WT mice [73% (8/11)] developed aneurysmal dilatations in the cerebral arteries without evidence of SAH. In contrast, half of mPGES-1 deficient mice died or showed signs of SAH during follow-up (P&lt;0.05). Postmortem analysis showed SAH in 56% (9/16) of mPGES-1 deficient mice. Unruptured aneurysms with no evidence of SAH were found in 25% (4/16) of mPGES-1 deficient mice. Subcutaneous infusion of PGE2 (1 mg/kg/d) to mPGES-1 KO mice did not appear to attenuate mortality or intracranial aneurysm rupture (p&gt;0.05). Another group of mPGES-1 KO mice were treated with aspirin (6 mg/kg/d) or vehicle (water) by gavage since day 2 after surgery. Mortality caused by symptomatic aneurysm rupture was significantly reduced in aspirin (25% (3/12) vs vehicle (58% (7/12)) treated mPGES-1 KO mice (P&lt;0.05). In conclusion, we found that genetic deficiency of mPGES-1 is associated with increased aneurysm rupture (p&lt;0.01) and mortality (p&lt;0.05) in a mouse model of intracranial aneurysms. Aspirin treatment significantly reduced mortality in mPGES-1 KO mice (P&lt;0.05). Therefore, mPGES-1 and aspirin appear to play a protective role against rupture of intracranial aneurysms in mice.

Reduced T Cell–Dependent Humoral Immune Response in Microsomal Prostaglandin E Synthase-1 Null Mice Is Mediated by Nonhematopoietic Cells

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of PGH2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis compared with wild-type (WT) mice associated with a marked reduction in Abs to type II collagen. In this study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum Abs in response to immunization with the T cell-dependent (TD) Ag DNP-keyhole limpet hemocyanin. Immunization with the T cell-independent type 1 Ag trinitrophenyl-LPS or the T cell-independent type 2 Ag DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleen of mPGES-1 null and WT mice were similar after immunization with DNP-keyhole limpet hemocyanin. To determine whether the effect of mPGES-1 and PGE2 was localized to hematopoietic or nonhematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in nonhematopoietic cells was the critical factor for reduced TD Ab production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of nonhematopoietic/mesenchymal stromal cells play a key role in TD humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation.

Abstract 83: Bone Marrow Derived Cells Do Not Mediate Reductions in Cholesterol, Atherosclerosis and Adiposity in Microsomal Prostaglindin E Synthase 1 Deficient Mice Fed High Fat Diets Enriched in Cholesterol

Microsomal prostaglandin E 2 synthase-1 (mPGES-1) catalyzes the conversion of COX-2 generated PGH 2 to PGE 2 and is the predominate source of PGE 2 during and inflammatory response. We and others have demonstrated that mPGES-1 deficiency attenuates atherosclerosis in mice on a mixed background. The present study investigated the effect of mPGES-1 deficiency on atherosclerosis in C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice. mPGES-1 deficiency attenuated atherosclerosis in LDLr-/- mice fed either a low fat (LF) (P = 0.02) or high fat (HF) (P = 0.0026) diet enriched with cholesterol, or a western diet (P = 0.02) for 17 weeks. mPGES-1 deficiency attenuated weight gain and cholesterol concentrations in mice fed a western (P = 0.004 and P &lt; 0.05; respectively) or HF diet (P = 0.01 and P = 0.012, respectively). However, body weight and cholesterol concentrations were not different in mice fed the LF diet. These data suggest that different mechanisms mediate the reduction in atherosclerosis in mPGES-1 deficient mice fed LF and HF diets. To determine if mPGES-1 deficiency in macrophages contributed to the reduction in atherosclerosis in mice fed HF diets, 4 groups of chimeric mice were generated. Four weeks post bone marrow cell transplant (BMT) mice were fed a western diet. BMT attenuated weight gain in all groups of chimeric mice; however, weight gain was not different between any of the groups. BMT decreased atherosclerotic lesion formation 10 fold in all groups of mice. Neither bone marrow cell specific deficiency of mPGES-1 (KO&gt;WT) or mPGES-1 specific expression in bone marrow derived cells (WT&gt;KO) had an effect on lesion formation compared to WT&gt;WT or KO&gt;KO mice. Cholesterol concentrations were decreased in KO&gt;KO and WT&gt;KO mice compared to WT&gt;WT (P &lt; 0.01) and KO&gt;WT (P&lt; 0.05) mice. These data suggest that mPGES-1 expression in bone marrow derived cells does not contribute to the development of atherosclerosis. Moreover, these data suggest that prostanoids may play a role in hepatic cholesterol homeostasis in mice fed HF diets enriched in cholesterol thereby contributing to atherosclerotic lesion formation. Moreover, these data provide further evidence that prostanoids play a role in regulating the accumulation of diet-induced adiposity.

Abstract 295: Microsomal Prostaglandin E Synthase 1 Deficiency Attenuates Diet-Induced Obesity and Promotes the Formation of Brite Adipose Tissue

Mice deficient in adipocyte specific phospholipases A2 have a marked reduction in prostaglandin E2 (PGE2) levels and are resistant to the development of diet-induced obesity. Clinical data suggest that obesity is a chronic low grade inflammatory disease, characterized by the influx of inflammatory cells into the adipose tissue. During a chronic inflammatory state, microsomal prostaglandin E synthase-1 (mPGES-1) is the primary source of PGE2. We have previously demonstrated that mice deficient in mPGES-1 (KO) have a marked reduction in body weight gain and adiposity compared to littermate controls (WT) fed a high fat (HF) diet with a concomitant reduction in urinary PGE2 concentrations and an increase in urinary PGI2 concentrations. The reduction in weight gain is not for accounted by alterations in food intake or locomotor activity. However, resting metabolic rate, measured by indirect calorimetry, was increased in KO mice compared to WT fed a HF diet. Moreover, body temperature was also increased in KO mice compared to WT mice (37.0 ± 0.2 vs 35.8 ± 0.2; P &lt; 0.05) fed a HF diet. Taken together these data suggest that mPGES-1 deficiency increases energy expenditure in response to feeding a HF diet. Analysis of white adipose tissue (WAT) depots demonstrated an increase in number of smaller adipocytes per unit area in the KO mice compared to WT mice. The WAT from KO mice also had a marked decrease in triglyceride content, F4/80 staining and CD86 staining with a concomitant increase in CD206 staining suggesting an attenuation in macrophage recruitment into the WAT as well as an M2 phenotype. Additionally, COX-2 and UCP-1 and PPAR-γ expression were increased in WAT depots with a concomitant localization of multi-locular adipocytes in WAT depots, demonstrating the presence of brown adipocytes in WAT depots in KO mice fed a HF diet. These data suggest that the reduction in body weight gain in the KO mice may be due an increase in thermogenesis mediated by the formation of brite adipose tissue in WAT depots.

Microsomal prostaglandin E synthase-1 is involved in multiple steps of colon carcinogenesis

Accumulating evidence indicates that cyclooxygenase (COX)-2-derived prostaglandin (PG) E(2) is involved in the development of various tumors, including colorectal cancer. However, the precise contribution of microsomal PGE synthase (mPGES)-1, a terminal enzyme that acts downstream of COX-2 in the PGE(2)-biosynthetic pathway, to multiple processes of tumor development is not yet fully understood. Here, we show the pro-tumorigenic role of mPGES-1 in chemical carcinogen-induced colon carcinogenesis and intrasplenic tumor transplantation models. Genetic deletion of mPGES-1 significantly reduced both the total number and size of colorectal polyps at 18 weeks after azoxymethane administration with reduced nuclear translocation of β-catenin, altered expression profiles of chemokines/cytokines and increased production of antitumorigenic PGs, prostaglandin D(2) and prostacyclin in tumor tissues. At an early stage (6 weeks), mPGES-1 deficiency significantly reduced the number of aberrant crypt foci, while its transgenic overexpression increased the number. Furthermore, the growth of intrasplenically transplanted tumor cells was suppressed in mPGES-1 knockout (KO) mice. Co-culture of tumor cells with bone marrow-derived macrophages (BM-MΦs) isolated from wild-type (WT) mice resulted in the induction of mPGES-1 in BM-MΦs and increased the growth of tumor cells in vitro, whereas mPGES-1-null BM-MΦs failed to facilitate tumor growth. The adoptive transfer of WT BM-MΦs into mPGES-1 KO mice restored the growth of transplanted tumor cells, indicating that mPGES-1 in MΦs is important for the growth of adjacent tumor cells. Taken together, our findings suggest that the inhibition of mPGES-1 is an alternative therapeutic target for colorectal and possibly other cancers.

The Impact of Microsomal Prostaglandin E Synthase 1 on Blood Pressure Is Determined by Genetic Background

Prostaglandin (PG)E(2) has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. Although microsomal PGE synthase (mPGES)1 is the only genetically verified PGE synthase, results of previous studies examining the consequences of mPGES1 deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1(-/-) mice on 2 distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1(-/-) mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1(-/-) animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1(-/-) and WT mice developed mild hypertension of similar magnitude, whereas 129-mPGES1(-/-) mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1(-/-) animals. In WT mice of both strains, the increase in urinary excretion of PGE(2) with angiotensin II was attenuated in mPGES1(-/-) animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1(-/-) mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.

Genetic Deletion of mPGES-1 Suppresses Intestinal Tumorigenesis

Elevated levels of prostaglandin E(2) (PGE(2)) are often found in colorectal cancers. Thus, nonsteroidal anti-inflammatory drugs, including selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, their long-term use is restricted by the occurrence of adverse events believed to be associated with a global reduction in prostaglandin production. In the present study, we evaluated the chemopreventive efficacy of targeting the terminal synthase microsomal PGE(2) synthase 1 (mPGES-1), which is responsible for generating PGE(2), in two murine models of intestinal cancer. We report for the first time that genetic deletion of mPGES-1 in Apc-mutant mice results in marked and persistent suppression of intestinal cancer growth by 66%, whereas suppression of large adenomas (>3 mm) was almost 95%. This effect occurred despite loss of Apc heterozygosity and beta-catenin activation. However, we found that mPGES-1 deficiency was associated with a disorganized vascular pattern within primary adenomas as determined by CD31 immunostaining. We also examined the effect of mPGES-1 deletion on carcinogen-induced colon cancer. The absence of mPGES-1 reduced the size and number of preneoplastic aberrant crypt foci (ACF). Importantly, mPGES-1 deletion also blocked the nuclear accumulation of beta-catenin in ACF, confirming that beta-catenin is a critical target of PGE(2) procarcinogenic signaling in the colon. Our data show the feasibility of targeting mPGES-1 for cancer chemoprevention with the potential for improved tolerability over traditional nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors.

Association of microsomal prostaglandin E synthase 1 deficiency with impaired fracture healing, but not with bone loss or osteoarthritis, in mouse models of skeletal disorders

Prostaglandin E synthase (PGES) functions as the terminal enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)) and is a potent regulator of bone and cartilage metabolism. Among the 3 isozymes of PGES, microsomal PGES-1 (mPGES-1) is known to play the most critical role in the production of PGE(2) in pathophysiologic events. This study investigated the roles of mPGES-1 under normal physiologic and pathophysiologic conditions in the skeletons of mPGES-1-deficient (mPGES-1(-/-)) mice. Skeletons of mPGES-1(-/-) mice and their wild-type littermates were compared by radiologic and histologic analyses. Four models of skeletal disorders were created: bone loss induced by ovariectomy, bone loss induced by hind limb unloading, osteoarthritis (OA) induced by instability in the knee joint, and bone fracture by osteotomy at the tibial midshaft. Expression of the PGES enzymes was examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction. The cellular mechanism of fracture healing was examined in ex vivo cultures of costal cartilage chondrocytes. Microsomal PGES-1(-/-) mice had unaffected skeletal phenotypes under normal physiologic conditions. In the bone fracture model, fracture healing was impaired by the mPGES-1 deficiency, with half of the mice remaining in a non-bone union state even after 21 days; normal fracture healing was restored by adenoviral reintroduction of mPGES-1. The other skeletal disorders were not affected by the mPGES-1 deficiency. In vivo and ex vivo analyses revealed an impaired proliferation of chondrocytes in cartilage with the mPGES-1 deficiency, at an early stage of fracture healing. In these mouse models of skeletal disorders, mPGES-1 was indispensable for bone repair through chondrocyte proliferation, but was not essential for the skeleton under normal physiologic conditions, nor did it play a role in the pathophysiologic conditions of bone loss due to ovariectomy, bone loss due to unloading, or stress-induced OA.

Hydrogen peroxide stimulates chloride secretion in primary inner medullary collecting duct cells via mPGES-1-derived PGE2

We investigated the role and mechanism of H2O2 in regulation of NaCl transport in primary inner medullary collecting duct (IMCD) cells. IMCD cells were isolated from wild-type mice and grown onto semipermeable membranes, and short-circuit current (Isc) was determined by Ussing chamber. Exposure of IMCD cells to H2O2 at a range of 100-300 microM caused a rapid increase in Isc in a time- and dose-dependent manner. This increase was almost abolished by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitors diphenylamine-2-carboxylic acid (DPC) and CFTR inhibitor-172. In contrast, the magnitude of stimulation was unaffected by the epithelial Na+ channel (ENaC) inhibitor amiloride. The H2O2-induced Cl(-) secretion was significantly inhibited by indomethacin, as well as by microsomal PGE synthase-1 (mPGES-1) deficiency. Like H2O2, PGE2 treatment induced a twofold increase in Isc that was reduced by the protein kinase A (PKA) inhibitors H-89 and KT5720. These data suggest that H2O2 stimulates CFTR Cl(-) channel-mediated Cl(-) secretion through cyclooxygenase- and mPGES-1-dependent release of PGE2 and subsequent activation of PKA.