EP4 Expression Research Articles

Prostaglandin receptor EP3 mediates growth inhibitory effect of aspirin through androgen receptor and contributes to castration resistance in prostate cancer cells

Although numerous epidemiological studies show aspirin to reduce risk of prostate cancer, the mechanism of this effect is unclear. Here, we first confirmed that aspirin downregulated androgen receptor (AR) and prostate-specific antigen in prostate cancer cells. We also found that aspirin upregulated prostaglandin receptor subtype EP3 but not EP2 or EP4. The EP3 antagonist L798106 and EP3 knockdown increased AR expression and cell proliferation, whereas the EP3 agonist sulprostone decreased them, indicating that EP3 affects AR expression. Additionally, EP3 (PTGER3) transcript levels were significantly decreased in human prostate cancer tissues compared with those in normal human prostate tissues, suggesting that EP3 is important to prostate carcinogenesis. Decreased EP3 expression was also seen in castration-resistant subtype CxR cells compared with parental LNCaP cells. Finally, we found that aspirin and EP3 modulators affected prostate cancer cell growth. Taken together, aspirin suppressed LNCaP cell proliferation via EP3 signaling activation; EP3 downregulation contributed to prostate carcinogenesis and to progression from androgen-dependent prostate cancer to castration-resistant prostate cancer by regulating AR expression. In conclusion, cyclooxygenases and EP3 may represent attractive therapeutic molecular targets in androgen-dependent prostate cancer.

Lymphocytic Colitis Is Associated with Increased Pro-Inflammatory Cytokine Profile and Up Regulation of Prostaglandin Receptor EP4

Microscopic colitis (MC) is comprised of two entities, lymphocytic (LC) and collagenous colitis. Up to 20% of patients with chronic diarrhea that have a normal appearing colonoscopy will be diagnosed with MC. Since MC was first described less than 40 years ago, little is known about the mechanisms involved in disease pathogenesis. Nonsteroidal anti-inflammatory drugs are associated with an increased risk of MC and some reports suggest a dysregulation in prostaglandin production. Recent genome wide screens have found an association between prostaglandin receptor EP4 expression and inflammatory bowel disease; however, EP4 expression has never been studied in MC. The aim of this study was to assess colonic mucosal inflammatory cytokine profiles in patients with LC and to assess expression of the prostaglandin receptor EP4. Colonic mucosal biopsies were obtained from patients undergoing colonoscopy for investigation of diarrhea and in those undergoing colon cancer screening. Following histological assessment, expression of cytokines and the prostaglandin receptor EP4 was analyzed using real-time reverse transcriptase-PCR and immunohistochemistry. Patients with LC had markedly increased mRNA expression for TNF-α, IFN-γ and IL-8 compared to normal controls (p<0.001). No significant differences were noted for IL-1β, IL-4, IL-10 or IL-12/23. Interestingly, those with LC had increased EP4 receptor expression, which positively correlated with increased TNF-α expression. This is the first report to demonstrate that LC is associated with increased TNF-α, INF-γ and IL-8 concurrent with a marked up-regulation of EP4. These findings add to our knowledge on the pathogenesis of LC and may give rise to possible new therapeutic and/or diagnostic tools in the management of MC.

Significance of Divergent Expression of Prostaglandin EP4 and EP3 Receptors in Human Prostate Cancer

PGE2 has been implicated in prostate cancer tumorigenesis. We hypothesized that abnormal prostaglandin receptor (EPR) expression may contribute to prostate cancer growth. Twenty-six archived radical prostatectomy specimens were evaluated by immunohistochemistry (IHC) and Western blotting for the expression of EP1, EP2, EP3, and EP4. As a corollary, EPR expression in one normal (PZ-HPV7) and four prostate cancer cell lines (CA-HPV10, LNCaP, PC3, and Du145) were assessed by Western blotting. Prostate cancer and normal cell growth were compared in vitro after EPR blockade, siRNA EPR knockdown, or overexpression. EP1, EP2, EP3, and EP4 receptors were detected by IHC in all areas of benign tissue within the clinical prostate cancer specimens. In areas of prostate cancer, EP4 and EP2 were overexpressed in 85% (22 of 26) and 75% (18 of 24) and EP3 expression was reduced in all (26 of 26, 100%) specimens (P < 0.05 vs. benign tissue). EP1 showed no specific differential expression pattern. Increased EP4 and reduced EP3 was confirmed by Western blotting in fresh clinical specimens and in prostate cancer cell lines (CA-HPV10, LNCaP, PC3, and Du145) compared with the normal prostate cell line (PZ-HPV7). EP2 and EP4 siRNA knockdown resulted in reduced in vitro growth and metastasis-related gene expression (MMP9 and Runx2) of prostate cancer lines, and in vitro migration was inhibited by EP4 antagonists. As a corollary, EP3-overexpressing PC3 cells displayed impaired growth in vitro. Human prostate cancer is associated with EP4 and EP2 overexpression and reduced EP3 expression. These data suggest that targeting specific EPR may represent a novel therapeutic approach for prostate cancer.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up‐regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin‐1 receptor antagonist‐deficient mice

The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. The levels of IL-1α, IL-1β, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A.actinomycetemcomitans LPS were significantly increased by approximately 4- (p<0.05), 5- (p<0.05), 1.3- (p<0.05) and 6- (p<0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p<0.05), 1.6- (p<0.05), 2.5- (p<0.05), 1.6- (p<0.05) and 1.9- (p<0.05) fold, respectively, in IL-1Ra KO mice stimulated with A.actinomycetemcomitans LPS compared with WT mice. IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Abstract 3862: Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer.

Abstract Prostaglandin E2 (PGE2), the chief cyclooxygenase-2 enzyme (COX-2) product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways with EP1 coupled to calcium mobilization and PKC. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with EP1 expression. Pharmacologic antagonism and reduction of EP1 expression increased metastatic capacity in our murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. We have also identified the presence of a variant EP1 (EP1v) transcript. EP1v has been identified previously in murine mast cell line MC/9 and rat uterus but not in malignant cells. EP1v has a pattern of mRNA expression different than full-length EP1. Compared to the cell line 410, EP1v expression is slightly increased in 410.4 and decreased in 66.1 cell lines. In order to determine the underlying mechanism in which EP1 suppresses metastasis, a metastasis gene array was performed comparing gene expression in EP1-vector, and EP1-silenced 66.1 cell lines and several candidate genes were identified including Fn1, whose protein is altered during epithelial-to-mesenchymal transition. qPCR analysis of Fn1 expression in EP1-silenced and overexpression cell lines revealed an inverse relationship between EP1 and Fn1 expression. A decrease in EP1 expression led to a 4-5 fold increase in Fn1 expression; whereas, increased EP1 expression resulted in a 0.50 fold decrease in Fn1 expression. Like full length EP1, overexpression of EP1v also resulted in a decrease in Fn1 expression. Bioinformatic analysis of the EP1 gene identified several CpG islands. DNA methylation analysis revealed hypermethylation of the CpG island nearest to the promoter in normal, non-metastatic and metastatic murine mammary tumor cell lines. In the quest to identify a clinically relevant strategy to increase expression of this protective receptor, we treated 410.4 and 66.1 cells with demethylating agent 5-azacytidine which resulted in an increase in EP1 expression. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2013-3862

Abstract 524: Cyclooxygenase pathway in ovarian cancer.

Abstract Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxygenases, specifically COX1 and COX2, are frequently overexpressed in malignancies and lead to the synthesis of prostaglandins. A particularly important prostaglandin, PGE2 is exported from the cell into the extracellular milieu by the multi-drug resistance protein 4 (MRP4) transporter where it acts in a paracrine and autocrine manner by activating a family of G-protein coupled receptors (EP1-4). PGE2 is transported into the cell by the prostaglandin transporter (PGT) and catabolized by 15-prostaglandin dehydrogenase (15-PGDH). It has been previously shown that COX1, rather than COX2, is overexpressed in ovarian cancer. Little is known about the role of other members of this pathway, including EP receptors, PGT, MRP4, and 15-PGDH, in this disease. Methods: HOSE (benign immortalized ovarian epithelial cells) and two ovarian cancer cell lines, SKOV3 and OVCAR3, were grown under standard conditions. Cells were harvested and RNA and protein were isolated. Quantitative RT-PCR was done using the SYBR Green protocol to examine the expression of members of the cyclooxygenase pathway and expressed relative to HOSE cells. Results: In both ovarian cancer cell lines relative to HOSE, expression of COX2, PGT, EP4, and EP1 was decreased. In SKOV3 cells, COX1 and 15-PGDH expression was decreased but MRP4 levels were comparable to levels detected in HOSE cells. In OVCAR3 cells, COX1 and 15-PGDH expression levels were increased and MRP4 was decreased relative to HOSE cells. PGE2 levels in media will be measured to determine the net effect of these changes. Protein levels of the members of the cyclooxygenase pathway will be determined by western blot. Conclusions: It is clear that several members of the cyclooxygenase pathway are expressed differently in malignant versus normal ovarian cells. Studies in progress will determine the functional significance of these differences. Citation Format: Gautam G. Rao, Tyler Kochel, Namita Kundu, Jocelyn Reader, Amy Fulton. Cyclooxygenase pathway in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2013-524

The Cyclooxygenase-2–Prostaglandin E2Pathway Maintains Senescence of Chronic Obstructive Pulmonary Disease Fibroblasts

Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by the irreversible loss of replicative capacity associated with the secretion of inflammatory mediators. However, the mechanisms of this phenomenon remain poorly defined. The aim of this study was to analyze the role of prostaglandin E2 (PGE2), a prostaglandin known to be increased in COPD lung fibroblasts, in inducing senescence and related inflammation in vitro in lung fibroblasts and in vivo in mice. Fibroblasts were isolated from patients with COPD and from smoker and nonsmoker control subjects. Senescence markers and inflammatory mediators were investigated in fibroblasts and in mice. Lung fibroblasts from patients with COPD exhibited higher expression of PGE2 receptors EP2 and EP4 as compared with nonsmoker and smoker control subjects. Compared with both nonsmoker and smoker control subjects, during long-term culture, COPD fibroblasts displayed increased senescent markers (increased senescence associated-β galactosidase activity, p16, and p53 expression and lower proliferative capacity), and an increased PGE2, IL-6, IL-8, growth-regulated oncogene (GRO), CX3CL1, and matrix metalloproteinase-2 protein and cyclooxygenase-2 and mPGES-1 mRNA expression. Using in vitro pharmacologic approaches and in vivo experiments in wild-type and p53(-/-) mice we demonstrated that PGE2 produced by senescent COPD fibroblasts is responsible for the increased senescence and related inflammation. PGE2 acts either in a paracrine or autocrine fashion by a pathway involving EP2 and EP4 prostaglandin receptors, cyclooxygenase-2-dependent reactive oxygen species production and signaling, and consecutive p53 activation. PGE2 is a critical component of an amplifying and self-perpetuating circle inducing senescence and inflammation in COPD fibroblasts. Modulating the described PGE2 signaling pathway could provide a new basis to dampen senescence and senescence-associated inflammation in COPD.

Wnt5a attenuates Wnt/β‐catenin signalling in human dermal papilla cells

Findings of recent studies have demonstrated modulation of Wnt/β-catenin signalling by Wnt5a, which is highly expressed in hair follicular dermal papilla (DP) in vivo. Here, we investigated the question of whether Wnt5a can affect canonical Wnt/β-catenin signalling in DP cells. Treatment with Wnt5a resulted in attenuation of Wnt3a-mediated elevation of β-catenin signalling, which was increased by Wnt5a siRNA transfection in cultured DP cells, as examined by reporter assay. In addition, treatment with Wnt5a resulted in repressed Wnt3a-mediated expression of Axin2, EP2 and LEF1 in cultured DP cells, whereas Wnt5a siRNA transfection resulted in increased Wnt3a-mediated expression of the genes in isolated DPs of cultured hair follicles. Moreover, treatment with Wnt5a resulted in attenuation of Wnt3a-mediated accumulation of β-catenin in the nucleus in DP cells. Our data strongly suggest that Wnt5a acts as an autocrine factor and attenuates canonical Wnt signalling pathway in human DP cells.

The Prostaglandin E2 Receptor, EP2, Is Upregulated in the Dorsal Root Ganglion After Painful Cervical Facet Joint Injury in the Rat

This study implemented immunohistochemistry to assay prostaglandin E2 (PGE2) receptor EP2 expression in the dorsal root ganglion (DRG) of rats after painful cervical facet joint injury. To identify if inflammatory cascades are induced in association with cervical facet joint distraction-induced pain by investigating the time course of EP2 expression in the DRG. The cervical facet joint is a common source of neck pain, and nonphysiological stretch of the facet capsular ligament can initiate pain from the facet joint via mechanical injury. PGE2 levels are elevated in painful inflamed and arthritic joints, and PGE2 sensitizes joint afferents to mechanical stimulation. Although in vitro studies suggest that the EP2 receptor subtype contributes to painful joint disease, the EP2 response has not been investigated for any association with painful mechanical joint injury. Separate groups of male Holtzman rats underwent either a painful cervical facet joint distraction injury or sham procedure. Bilateral forepaw mechanical allodynia was assessed, and immunohistochemical techniques were used to quantify EP2 expression in the DRG at days 1 and 7. Facet joint distraction induced mechanical allodynia that was significant (P < 0.024) at all time points. Painful joint injury also significantly elevated total EP2 expression in the DRG at day 1 (P = 0.009), which was maintained at day 7 (P < 0.001). Neuronal expression of EP2 in the DRG was only increased over sham levels at day 1 (P = 0.013). Painful cervical facet joint distraction induces an immediate and sustained increase of EP2 expression in the DRG, implicating peripheral inflammation in the initiation and maintenance of facet joint pain. The transient increase in neuronal EP2 suggests, as in other painful joint conditions, that after joint injury nonneuronal cells may migrate to the DRG, some of which likely express EP2.

Involvement of the prostaglandin E receptor EP2 in paeoniflorin-induced human hepatoma cell apoptosis

Prostaglandin E2 (PGE2) has been shown to play an important role in tumor development and progression. PGE2 mediates its biological activity by binding any one of four prostanoid receptors (EP1 through EP4). The present study was designed to determine the role of the EP2 receptor during the proliferation and apoptosis of human HepG2 and SMMC-7721 hepatoma cell lines and the effect of paeoniflorin, a monoterpene glycoside. The proliferation of HepG2 and SMMC-7721 cells was determined by methyl thiazolyl tetrazolium after exposure to the selective EP2 receptor agonists butaprost and paeoniflorin. Apoptosis of HepG2 and SMMC-7721 cells was also quantified by flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodide staining. The expression levels of Bcl-2 and Bax were quantified by western blotting and immunohistochemistry. The expression of the EP2 receptor and cysteine-aspartic acid protease (caspase)-3 was determined by western blotting. Butaprost significantly increased proliferation in HepG2 and SMMC-7721 cells. Paeoniflorin significantly inhibited the proliferation of HepG2 and SMMC-7721 cells stimulated by butaprost at multiple time points (24, 48, and 72 h). Paeoniflorin induced apoptosis in HepG2 and SMMC-7721 cells, which was quantified by annexin-V and propidium iodide staining. Our results indicate that the expression of the EP2 receptor and Bcl-2 was significantly increased, whereas that of Bax and cleaved caspase-3 was decreased in HepG2 and SMMC-7721 cells after stimulation by butaprost. Paeoniflorin significantly decreased the expression of the EP2 receptor and Bcl-2 and increased Bax and caspase-3 activation in HepG2 and SMMC-7721 cells on addition of butaprost. Our results show that the PGE2 receptor subtype EP2 may play a vital role in the survival of both HepG2 and SMMC-7721 cells. Paeoniflorin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in hepatocellular carcinoma cells by downregulating EP2 expression and also increased the Bax-to-Bcl-2 ratio, thus upregulating the activation of caspase-3.

Genetic susceptibility for Stevens-Johnson syndrome/Toxic epidermal necrolysis with mucosal involvements

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute inflammatory vesiculobullous reactions of the skin and mucous membranes including oral cavity and ocular surface. Single nucleotide polymorphism (SNP) association analysis of SJS/TEN patients with severe mucosal involvements (MI) revealed that TLR3 SNPs and IL4R SNP Gln551Arg were significantly associated with SJS/TEN with MI. We also found that PTGER3 SNPs were associated with the SJS/TEN using genome-wide association study (GWAS). The expression of EP3 (protein of PTGER3 gene ) was greatly reduced in the conjunctival epithelium of the SJS/TEN patients compared to the controls. About 80% of our SJS/TEN patients with MI had used cold medications before the onset of their disease, and cold medicines including non-steroid anti-inflammatory drugs (NSAIDs) could inhibit the production of the EP3 ligand PGE2. Thus, EP3 may contribute to the development of SJS/TEN with MI. We also found that a combination of TLR3 and PTGER3 PTGER3 PTGER3 PTGER3 SNPs could raise the genetic susceptibility of SJS/TEN with MI, and that there were functional interactions between TLR3 and EP3, the protein of PTGER3. This suggests that a lack of balance between TLR3 and EP3 may trigger mucosal inflammation at sites such as the ocular surface. Elsewhere we documented that in Japanese patients HLA-A*0206 is strongly associated with the SJS/TEN. We also reported multiplicative interaction(s) between HLA-A*0206 and TLR3 SNPs in patients with the SJS/TEN. Together, our observations suggest that not only environmental- but also genetic factors, including epistatic interactions, play a role in an integrated etiology of SJS/TEN with MI.

Fluid flow shear stress upregulates prostanoid receptor EP2 but not EP4 in murine podocytes

Podocytes in the glomerular filtration barrier regulate the passage of plasma proteins into urine. Capillary pressure and ultrafiltration impact the structure and function of podocytes. The mechanism of podocyte injury by fluid flow shear stress (FFSS) from hyperfiltration in chronic kidney disease (CKD) is not completely understood. Recently, we demonstrated increased synthesis of prostaglandin E2 in podocytes exposed to FFSS. Here, we determine the effect of FFSS on prostanoid receptors EP1–EP4 in cultured podocytes and in Os/+ mouse kidney, a model of hyperfiltration. Results of RT-PCR, qRT-PCR, immunoblotting and immunofluorescence studies indicate that cultured podocytes express EP1, EP2 and EP4 but not EP3. FFSS resulted in upregulated expression of only EP2 in podocytes. Kidney immunostaining showed significantly increased expression of EP2 in Os/+ mice compared with littermate controls. These novel results suggest that EP2 may be responsible for mediating podocyte injury from hyperfiltration-induced augmented FFSS in CKD.

Prostaglandin E2 receptors have differential effects on Leishmania major infection

Through their receptors, prostaglandins play crucial roles in various infections. Although prostaglandin E₂ (PGE₂) is implicated as a susceptibility factor in Leishmania infection, the relative contributions of its four receptors--EP1, EP2, EP3 and EP4--to this infection remain unknown. We report that Leishmania major infection of BALB/c-derived peritoneal macrophages up-regulated EP1 and EP3 expressions but down-regulated EP2 and EP4 expressions. EP2 and EP4 agonists reduced parasite load, but EP1 and EP3 agonists increased parasite load in macrophages in vitro. Agonists of EP2 and EP4, antagonists of EP1 and EP3, or lentivirally expressed EP1-shRNA and EP3-shRNA significantly reduced parasite burden in susceptible BALB/c mice. These novel data suggest differential regulation and counteractive functions of EP receptor subsets.

Pretreatment with paricalcitol attenuates inflammation in ischemia-reperfusion injury via the up-regulation of cyclooxygenase-2 and prostaglandin E2

The effect of paricalcitol on renal ischemia-reperfusion injury (IRI) has not been investigated. We examined whether paricalcitol is effective in preventing inflammation in a mouse model of IRI, and evaluated the cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) pathways as a protective mechanism of paricalcitol. Paricalcitol (0.3 μg/kg) was administered to male C57BL/6 mice 24 h before IRI. Bilateral kidneys were subjected to 23 min of ischemia, and mice were killed 72 h after IRI. The effects of paricalcitol on renal IRI were evaluated in terms of renal function, tubular necrosis, apoptotic cell death, inflammatory cell infiltration and inflammatory cytokines. The effects of paricalcitol on COX-2, PGE2 and its receptors were investigated. Paricalcitol pretreatment improved renal function (decreased blood urea nitrogen and serum creatinine levels), tubular necrosis and apoptotic cell death in IRI-mice kidneys. The infiltration of inflammatory cells (T cells and macrophages), and the production of proinflammatory cytokines (RANTES, tumor necrosis factor-α, interleukin-1β and interferon-γ) were reduced in paricalcitol-treated mice with IRI. Paricalcitol up-regulated COX-2 expression, PGE2 synthesis and mRNA expression of receptor subtype EP4 in post-ischemic renal tissue. The cotreatment of a selective COX-2 inhibitor with paricalcitol restored functional injury and tubular necrosis in paricalcitol-treated mice with IRI. Our study demonstrates that paricalcitol pretreatment prevents renal IRI via the inhibition of renal inflammation, and the up-regulation of COX-2 and PGE2 is one of the protective mechanisms of paricalcitol in renal IRI.

E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling.

The experimental administration of PGE(2) for the treatment of asthma dampens clinical symptoms, and similar efficacy has been found in dust mite-induced hypersensitivity reactions in animal models. Here, we investigate the mechanism by which PGE(2) mediates suppression of MC degranulation. We find that the effect of PGE(2) on FcεRI-dependent MC degranulation varies from activating to suppressing, depending on the relative ratio of EP(2) to EP(3) expression on these cells with suppression evident only in cells having increased EP(2) to EP(3) expression. Consistent with a role for EP(2) in suppressing MC responses in vitro, we found that a selective EP(2) agonist, Butaprost, inhibited MC-mediated FcεRI-induced immediate hypersensitivity in a model of PCA. EP(2) engagement on MCs increased cAMP production and inhibited FcεRI-mediated calcium influx. In addition, it also decreased the extent of FcεRI-induced Fyn kinase activity, leading to decreased phosphorylation of key signaling molecules such as Gab2 and Akt. Treatment with an antagonist of cAMP or shRNA down-regulation of PKA (the principal intracellular target of cAMP) reversed the EP(2)-mediated inhibitory effect on MC degranulation and restored calcium influx and phosphorylation of Akt. Collectively, the findings demonstrate that EP(2) suppresses the Fyn-mediated signals that are central to FcεRI-dependent MC degranulation, suggesting that engagement of the EP(2) on MCs may be beneficial in dampening allergic responses.

S129 Decreased cAMP Production in Lung Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

RationaleIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with unknown aetiology and no effective therapy. Myofibroblasts are the primary effector cells in the pathogenesis of IPF and differentiation from...

Activation of Prostaglandin E Receptor 4 Triggers Secretion of Gut Hormone Peptides GLP-1, GLP-2, and PYY

Prostaglandins E1 and E2 are synthesized in the intestine and mediate a range of gastrointestinal functions via activation of the prostanoid E type (EP) family of receptors. We examined the potential role of EP receptors in the regulation of gut hormone secretion from L cells. Analysis of mRNA expression in mouse enteroendocrine GLUTag cells demonstrated the abundant expression of EP4 receptor, whereas expression of other EP receptors was much lower. Prostaglandin E1 and E2, nonselective agonists for all EP receptor subtypes, triggered glucagon like peptide 1 (GLP-1) secretion from GLUTag cells, as did the EP4-selective agonists CAY10580 and TCS2510. The effect of EP4 agonists on GLP-1 secretion was blocked by incubation of cells with the EP4-selective antagonist L161,982 or by down-regulating EP4 expression with specific small interfering RNA. Regulation of gut hormone secretion with EP4 agonists was further studied in mice. Administration of EP4 agonists to mice produced a significant elevation of plasma levels of GLP-1, glucagon like peptide 2 (GLP-2) and peptide YY (PYY), whereas gastric inhibitory peptide (GIP) levels were not increased. Thus, our data demonstrate that activation of the EP4 receptor in enteroendocrine L cells triggers secretion of gut hormones.

Paricalcitol Attenuates Ischemia-Reperfusion Injury in Mice Kidney

Ischemia-reperfusion injury (IRI) is unavoidable event in renal transplantation, causing the delayed graft function and increased immunogenicity. We investigated whether paricalcitol is renoprotective in a mouse model of IRI, and whether potential mechanism is related with modulation of renal inflammation and prostaglandin E2 (PGE2) synthesis. Paricalcitol (0.3 μ/Kg) was administered to male C57BL/6 mice 24 hours before IRI, and mice were killed at 72 hours after IRI. Treatment with paricalcitol decreased blood urea nitrogen levels, serum creatinine levels, tubular necrosis score. Paricalcitol decreased the number of TUNEL-positive cells and reduced the expression of apoptotic markers. The production of RANTES and tumor necrosis factor-α were reduced by paricalcitol and the infiltration of CD40-positive antigen-presenting cells were decreased in the paricalcitol-treated kidneys. Paricalcitol inhibited the production of Th1 cytokines interleukin (IL)-2 and interferon-γ, which was accompanied with decreased infiltration of T-cells and macrophages. The immunomodulatory effects of paricalcitol also affected the decreased production of Th2 cytokines IL-4 and IL-10. Paricalcitol induced the increased cyclooxygenase (COX)-2 expression and decreased the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) production, which lead to increased endogenous PGE2 synthesis. Of the PGE2 receptors, the expression of EP1 and EP4 were increased in the paricalcitol-treated kidneys and the activation of EP4 receptors increased the expression of survival kinase Akt. In conclusion, our study demonstrated that paricalcitol has a protective effect on renal IRI and that this effect is associated with inhibition of renal inflammatory infiltration, increased PGE2 synthesis and EP4 receptor activation.

Eicosanoids, Prostaglandins, and the Progression of Tuberculosis

Eicosanoids, Prostaglandins, and the Progression of Tuberculosis

PROSTAGLANFIN E RECEPTORS AS INFLAMMATORY THERAPEUTIC TARGETS FOR ATHEROSCLEROSIS

ObjectivesAtherosclerosis is currently considered to be a chronic inflammatory disease. Prostaglandin E receptors (EPs) are the G-protein-coupled receptors that respond to prostaglandin E2 (PGE2). Data has shown that PGE2 may...