Expression Of Prostaglandin E2 Receptors Research Articles

Role of COX-2/PGE2/EP4 Axis-induced Macrophage Functional Activation in NSCLC Development

Tumor microenvironment (TME) is one of the important factors in tumorigenesis and progression, in which tumor-associated macrophages (TAMs) play an important role in non-small cell lung cancer (NSCLC) progression. However, the mechanism of TAMs in NSCLC progression remains unclear, so this study aimed to investigate the role of TAMs in NSCLC progression and to find potential therapeutic targets. Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the expression of prostaglandin E2 receptor 4 (EP4) mRNA in NSCLC and normal lung tissues; the protein expression levels of cyclooxygenase-2 (COX-2), EP4, cluster of differentiation 86 (CD86), CD163 and CD31 were detected by immunohistochemistry (IHC) in 120 NSCLC tissues and 24 paracancerous tissues specimens. The nude mouse lung adenocarcinoma cell A549 and macrophage RAW264.7 co-transplanted tumor model was established. And the samples were collected by gavage with EP4 inhibitor E7046, and then stained with hematoxylin-eosin (HE), IHC, and immunofluorescence (IF), and then detected by Western blot for the epithelial mesenchymal transformation (EMT) of the tumor tissues of the nude mice in each group. Western blot was used to detect the expressions of EMT related protiens in each group of nude mice; full-length transcriptome sequencing was used to screen the key genes causing liver metastasis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. EP4 mRNA expression level in NSCLC tissues was generally lower than that in normal lung tissues (P<0.05); COX-2, EP4, CD163, CD31 proteins were differentially expressed in NSCLC tissues and adjacent tissues, and differences were observed in many clinicopathological parameters of NSCLC patients; RAW264.7 shortened the latency period of tumorigenesis of A549 and promoted the proliferation of tumors and liver metastasis of tumors, and E7046 could reduce tumor cell proliferation activity, tumor tissue vascular density and M2-type macrophage infiltration in nude mice; IF staining showed that macrophages were mainly distributed around the metastatic foci of tumors; Western blot results showed that compared with A549 alone transplantation group, the relative expression of E-cadherin protein in tumor tissues of mice in A549 and RAW264.7 co-transplantation group was significantly decreased, and the difference was statistically significant (P<0.05), while the relative expression of N-cadherin protein was up-regulated, but the difference was not statistically significant (P>0.05); the main pathways enriched in the differential genes of the full-length transcriptome were the PI3K-AKT and MAPK signaling pathways. During NSCLC development, the COX-2/PGE2/EP4 axis may promote tumor progression by inducing macrophage functional activation, and EP4 may be a potential new target for tumor immunotherapy. This study provides new perspectives and ideas for in-depth exploration of the mechanisms of NSCLC development, as well as a theoretical basis for the development of new therapeutic strategies for NSCLC.

Prostaglandin E2 induces ovulation in prepubertal mice

The objective of this study was to determine the ability of prostaglandin E2 (PGE2) to induce ovulation and expression of PGE2 receptor (EP2 and EP4) and COX genes (COX-1 and COX-2) in the ovary and pituitary of prepubertal mice. The positive control consisted of the application of 5 μg of gonadotropin-releasing hormone (GnRH, n = 29); the negative control applied 0.5 mL of phosphate buffered saline (PBS, n=31); the treatment tested the application of 250 μg of PGE2 (n = 29), making a total of 89 prepubertal mice (BALB/c). Mice were euthanized 14 to 15 h after treatments to detect ovulation and tissue collection. A Chi-square test was used to compare the proportion of animals ovulating. Gene expressions and number of ovulation were analyzed by one-way ANOVA and Tukey’s test was used to compare means among groups. A greater proportion of mice (P &lt; 0.001) ovulated after receiving GnRH (89.7%, 26/29) compared to PGE2 group (58.6%, 17/29). However, the proportion was higher compared to those treated with PBS (0%, 0/31). Ep2 gene expression in the pituitary was &gt; two-fold higher (P &lt; 0.05) in the PGE2 group compared to the PBS and GnRH groups. Further, PGE2 stimulated Cox1 (2.7 fold, P &lt; 0.05) while GnRH stimulated Cox2 expression (6.5 fold, P &lt; 0.05) in the pituitary when compared to the PBS group. In conclusion, our results support the hypothesis that PGE2 can induce ovulation in prepubertal mice with a concomitant increase in Ep2 and Cox1 gene expression in the pituitary gland.

PGE2 mediates hemocyte-spreading behavior by activating aquaporin via cAMP and rearranging actin cytoskeleton via Ca2+

Spreading behavior of hemocytes (= insect blood cells) is essential for cellular immune responses against various microbial pathogens. It is activated by prostaglandin E2 (PGE2) via its membrane receptor associated with secondary messenger, cAMP, in insects. This study observed an increase of calcium ion (Ca2+) level after an acute increase of cAMP induced by PGE2 treatment and clarified the intracellular signals underlying the hemocyte-spreading behavior. Inhibition of Ca2+ flux significantly impaired the hemocyte-spreading and subsequent cellular immune response, phagocytosis. The up-regulation of intracellular Ca2+ in response to PGE2 was dependent on cAMP because RNA interference (RNAi) of PGE2 receptor expression or inhibiting adenylate cyclase prevented Ca2+ mobilization. The up-regulation of Ca2+ was induced by inositol triphosphate (IP3) via its specific IP3 receptor. Furthermore, inhibition of ryanodine receptor impaired Ca2+ mobilization, suggesting Ca2+-induced Ca2+ release. However, the effective spreading behavior of hemocytes was dependent on both secondary messengers. Ca2+ signal stimulated by cAMP was required for activating small G proteins because RNAi treatments of small G proteins such as Rac1, RhoA, and Cdc42 failed to stimulate hemocyte-spreading. In contrast, aquaporin was activated by cAMP. Its activity was necessary for changing cell volume during hemocyte-spreading. These results indicate that PGE2 mediates hemocyte-spreading via cAMP signal to activate aquaporin and via Ca2+ signal to activate actin cytoskeletal rearrangement.

Cardiac injury modulates critical components of prostaglandin E2 signaling during zebrafish heart regeneration

The inability to effectively stimulate cardiomyocyte proliferation remains a principle barrier to regeneration in the adult human heart. A tightly regulated, acute inflammatory response mediated by a range of cell types is required to initiate regenerative processes. Prostaglandin E2 (PGE2), a potent lipid signaling molecule induced by inflammation, has been shown to promote regeneration and cell proliferation; however, the dynamics of PGE2 signaling in the context of heart regeneration remain underexplored. Here, we employ the regeneration-competent zebrafish to characterize components of the PGE2 signaling circuit following cardiac injury. In the regenerating adult heart, we documented an increase in PGE2 levels, concurrent with upregulation of cox2a and ptges, two genes critical for PGE2 synthesis. Furthermore, we identified the epicardium as the most prominent site for cox2a expression, thereby suggesting a role for this tissue as an inflammatory mediator. Injury also drove the opposing expression of PGE2 receptors, upregulating pro-restorative ptger2a and downregulating the opposing receptor ptger3. Importantly, treatment with pharmacological inhibitors of Cox2 activity suppressed both production of PGE2, and the proliferation of cardiomyocytes. These results suggest that injury-induced PGE2 signaling is key to stimulating cardiomyocyte proliferation during regeneration.

Prostaglandin E2, a postulated mediator of neurovascular coupling, at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance-sized human cerebral parenchymal arterioles.To achieve this goal PGE2-induced isotonic vasomotor responses were assessed in parenchymal arterioles isolated from fronto-temporo-parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined.In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10−8 to 10-6 mol/l) caused significant, endothelium-independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1-dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA and protein expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles.PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human patients.

Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial-mesenchymal transition participation in cardiac remodelling.

Long non‐coding RNAs (lncRNAs) have emerged as potent regulators of cardiac disease; however, the role of lncRNA in cardiac fibrosis remains partially understood. In this study, we identified a cardiac endothelial‐enriched lncRNA‐lnc000908, which was markedly up‐regulated in rats with cardiac fibrosis. In addition, the expression of prostaglandin E2 receptor 4 (EP4) was decreased in cardiac fibrosis. In vivo lnc000908 silencing by lentivirus increased the EP4 level, decreased endothelial‐mesenchymal transition (EndMT) and improved cardiac fibrosis and cardiac function. Consistently, the lnc000908 knockdown also up‐regulated EP4 and suppressed transforming growth factor‐beta (TGF‐β)‐induced EndMT in cardiac microvascular endothelial cells. In contrast, the lnc000908 overexpression by lentivirus decreased the EP4 level and induced EndMT. Of note, these pro‐ or anti‐EndMT effects were reversed by the EP4 overexpression or the EP4 antagonist AH‐23848, respectively. This study demonstrates that lnc000908 is a novel regulator of cardiac fibrosis by modulating the EP4 expression and EndMT.

Expression of prostaglandin E2 receptor 3 in the eyelid epidermis of patients with Stevens-Johnson syndrome/toxic epidermal necrolysis

Background/aimsIn a previous genome-wide association study of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) patients we reported the association between SJS/TEN and the prostaglandin E receptor 3 (PTGER3) gene, and that its...

Electroacupuncture for pain relief in labour inhibits spinal p38 MAPK-mediated prostaglandin E2 release and uterine prostaglandin E2 receptor expression in rats.

p38 mitogen-activated protein kinase (p38 MAPK) activation involves the release of prostaglandin E2 (PGE2) and hyperalgesia. We have previously reported that electroacupuncture (EA) relieves labour pain, but the potential mechanisms remain unclear. To observe the effects of EA on labour pain intensity, serum PGE2 levels and the p38 MAPK signalling pathway in rats during labour. Female rats copulated with male rats to induce pregnancy, and then received castor oil to trigger labour. During labour, rats remained untreated (Control group, n=30) or were treated with remifentanil (n=30) or EA at Jiaji (n=30) or SP6+LI4 (n=30), respectively. The warm water tail-flick test was used to assess labour pain. Serum PGE2 levels were measured by ELISA. Protein expression of prostaglandin E2 receptor (PGER2), p38 MAPK and phospholipase A2 (PLA2) were analysed by Western blotting, and mRNA levels were measured by real-time PCR. EA treatment at Jiaji or SP6+LI4 significantly relieved labour pain, decreased serum PGE2 levels and inhibited protein and gene expression of PGER2 in the myometrium. Moreover, EA reduced protein expression of PLA2 and p38 MAPK, and inhibited phosphorylation of p38 MAPK in the lumbar spinal cord but not in the cerebral grey matter. Additionally, EA markedly decreased mRNA levels of p38 MAPK in the lumbar spinal cord and significantly reduced PLA2-IV mRNA levels in both the lumbar spinal cord and cerebral grey matter. This study indicates that EA relieves labour pain through, at least in part, inhibition of spinal p38 MAPK-mediated PGE2 release and uterine PGER2 expression in rats.

Prostaglandin E2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles: implications for neurovascular coupling responses

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance‐sized human cerebral parenchymal arterioles.To achieve this goal PGE2‐induced isotonic responses were assessed in parenchymal arterioles isolated from fronto‐temporo‐parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined.In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10−8 to 10−6 mol/l) caused significant, endothelium‐independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1‐dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles. In contrast, rat vessels expressed predominantly EP1 receptors.PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human subjects.Support or Funding InformationThis work was supported by grants from the National Research, Development and Innovation Office to PT (NKFI‐FK123798) and AK (K108444), the Hungarian Academy of Sciences Bolyai Research Scholarship BO/00634/15 to PT, the PTE AOK‐KA 3/2016 04.01/F to PT, the ÚNKP‐18‐4‐PTE‐6 New National Excellence Program of the Ministry of Human Capacities, Higher Education Institutional Excellence Programme – Grant No. 20765‐3/2018/FEKUTSTRAT, EFOP‐3.6.2.‐16‐2017‐00008, GINOP‐2.3.2‐15‐2016‐00048 and GINOP‐2.3.3‐15‐2016‐00032 to PT and AB, the Hungarian National Brain Research Program Grant No. 2017‐1.2.1‐NKP‐2017‐00002 to AB; the Marie Curie Actions SMARTER 7th Framework Program of the European Union 606998 to NSz and AK, National Institute of Health R01‐AG055395, R01‐NS100782, R01‐AT006526 to ZU.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E2 (PGE2) is responsible for inflammatory symptoms. However, PGE2 also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE2 receptors, EP1–EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R–EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells.Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE2, alongside selective agonists of EP1–EP4 receptors, were assessed on LPS-induced TNF-α, IL-1β and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs.PGE2 and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE2 on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE2.This indicates that PGE2 suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.

Effect of sodium hydrosulfide on mRNA expression of prostaglandin E2 receptors in response to mucosal acidification and distention-induced gastric acid secretion in rats.

Objective(s):Prostaglandins have been shown to mediate the gastro-protective effect of sodium hydrosulfide (NaHS) but effect of NaHS on mRNA expression of prostaglandin E2 receptors (EP1, 3-4; EPs) has not been investigated. Therefore, this study designed to evaluate the effect of NaHS on mRNA expression of EPs receptors in response to mucosal acidification and distention-induced gastric acid secretion in rats.Materials and Methods:Fasted rats were randomly assigned into 4 groups (n=6/group). They were control, and NaHS-treated groups. To evaluate the effect of NaHS on mucosal mRNA expression of EPs receptors, the gastric mucosa exposed to stimulated gastric acid output and mucosal acidification. The pylorus sphincter catheterized for instillation of isotonic neutral saline or acidic solution. Ninety min after beginning the experiments, animals sacrificed and the gastric mucosa collected to determine the pH, mucus secretion and to quantify the mRNA expression of EPs receptors by quantitative real-time PCR.Results:present results showed that a) NaHS increased the mucus secretion, mRNA expression of EP3 and EP4 receptors in response to distention-induced expression; b) The mRNA expression of EP1 receptors increased while EP4 mRNA receptors decreased in response to mucosal acidification in NaHS-pretreated rats; and c) NaHS increased pH of gastric contents both in response to distention-induced gastric acid secretion and mucosal acidification.Conclusion:NaHS behaves in a different manner. It effectively only increased the pH of gastric contents to reinforce the gastric mucosa against a highly acidic solution but modulated both acid and mucus secretion when the rate of acid increase in the stomach was slower.

The role of the prostaglandin E2 receptors in vulnerability of oligodendrocyte precursor cells to death.

BackgroundActivity of cyclooxygenase 2 (COX-2) in mouse oligodendrocyte precursor cells (OPCs) modulates vulnerability to excitotoxic challenge. The mechanism by which COX-2 renders OPCs more sensitive to excitotoxicity is not known. In the present study, we examined the hypothesis that OPC excitotoxic death is augmented by COX-2-generated prostaglandin E2 (PGE2) acting on specific prostanoid receptors which could contribute to OPC death.MethodsDispersed OPC cultures prepared from mice brains were examined for expression of PGE2 receptors and the ability to generate PGE2 following activation of glutamate receptors with kainic acid (KA). OPC death in cultures was induced by either KA, 3′-O-(Benzoyl) benzoyl ATP (BzATP) (which stimulates the purinergic receptor P2X7), or TNFα, and the effects of EP3 receptor agonists and antagonists on OPC viability were examined.ResultsStimulation of OPC cultures with KA resulted in nearly a twofold increase in PGE2. OPCs expressed all four PGE receptors (EP1–EP4) as indicated by immunofluorescence and Western blot analyses; however, EP3 was the most abundantly expressed. The EP3 receptor was identified as a candidate contributing to OPC excitotoxic death based on pharmacological evidence. Treatment of OPCs with an EP1/EP3 agonist 17 phenyl-trinor PGE2 reversed protection from a COX-2 inhibitor while inhibition of EP3 receptor protected OPCs from excitotoxicity. Inhibition with an EP1 antagonist had no effect on OPC excitotoxic death. Moreover, inhibition of EP3 was protective against toxic stimulation with KA, BzATP, or TNFα.ConclusionTherefore, inhibitors of the EP3 receptor appear to enhance survival of OPCs following toxic challenge and may help facilitate remyelination.

Evidence for prostaglandin E2 receptor expression in the intramural ganglia of the guinea pig urinary bladder

Intramural ganglia are present in the bladder wall of several species including human, pig, and guinea-pig. It has been suggested that there is a network of intramural ganglia in the bladder of these species that may be part of a motor-sensory system and receive afferent input. Prostaglandins (PG) have been suggested to play a role in this afferent signalling mechanism. To investigate the distribution of the prostaglandin E2 receptors EP1 and EP2 in and around intramural ganglia of the guinea pig, bladders of 6 guinea pigs were dissected, and processed for immunohistochemistry. Sections were examined for prostaglandin E2 receptor EP1- and EP2-immuno-reactivity and co-stained for vimentin, a marker for interstitial cells (IC) and cyclo-oxygenase 1 (COX I), the enzyme responsible for PG synthesis.Immunoreactivities for EP1 and EP2 were found in intramural ganglion cells. These cells were observed in between muscle bundles and on, or close to the serosal surface of the bladder. Furthermore, COX I was present in interstitial cells close to ganglion cells, indicating the possibility of a local synthesis of prostaglandins near the ganglia. The co-staining of EP1 or EP2 with vimentin showed that processes of interstitial cells run through the ganglia, often encircling or ensheathing cells.Therefore, it can be concluded that there is a close relationship between the intramural ganglia and the network of interstitial cells in the muscular layers of the bladder. EP1 and EP2 receptors are expressed on the ganglia and this arrangement suggests that intramural ganglia are involved in (pre)processing afferent information.

Regional and temporal regulation of prostaglandin E2 receptor expression: a role for decidual activation in term labor (911.7)

Objective: To assess if expression of prostaglandin E2 receptors (EP1, EP2, EP3 and EP4) are spatially or temporally regulated in decidua with labour onset.Methods: Decidua biopsies from caesarean section deliveries, of labouring (TL) and non‐labouring (TNL) women and from both upper (US) and lower segment (LS) regions of the uterus were collected (n=12/group). Real‐time RT‐PCR was used to quantify levels of EP1‐4 gene expression.Results: All four EPs are expressed in US and LS decidua. Expression of EP1, EP2, and EP4 is higher in LS compared to US in TNL decidua, but did not change with labor initiation. In contrast, EP3 was significantly higher than the other EP receptors in both US and LS in TNL decidua. The EP3 mRNA levels decrease in LS but not US decidua with labor initiation.Conclusions: The presence of four EP receptor subtypes within the decidua in labouring and non‐labouring women suggests that prostaglandin E2 plays an active biological role during pregnancy and labour via differential EP interaction. Our observation that EP3 levels change with labor onset suggests that EP3 are the predominant mediators of PGE2 activity in the decidua at term. These findings on differential EP regulation in decidua with labour are important for the understanding of the underlying mechanisms that govern uterine contractility and quiescence.Grant Funding Source: Supported by Canadian Institute of Health Research and Alberta Innovates‐Health Solutions

Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells.

e21107 Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells Background: Only a small fraction of breast tumor cells, breast cancer-initiating cells (CSC), have the ability to initiate tumor growth and metastasis. Some characteristics of breast CSCs have been described in vitro by employing non-adherent CSC-enriching culture conditions, however, little is known regarding prostanoid signaling or prostaglandin E2 (PgE2) production in CSCs. In this study we measured cyclooxygenase-2 (COX-2) and prostaglandin E2 receptor expression in CSCs and assessed the effects of COX-2 and Ep4 inhibition on in vitro CSC spheroid formation and PgE2 production. Methods: CSCs were cultured in serum-free medium using ultra low attachment plates. Cells were treated with vehicle, a COX-2 inhibitor (Celecoxib), or an EP4 inhibitor (GW627368X) at concentrations of 0.1, 1, 5, 10, 50, or 100uM for 10 days. Spheroids were quantified using a Gelcount machine. Non-linear regression was used to calculate IC50 values. Western blots were performed using anti-COX-2, anti- EP1, 2, 3 and 4 receptor antibodies. Prostaglandin E2 production was measured using a PgE2 immunoassay kit. Results: COX-2 protein expression was observed for MDA-MB-231 and SUM149 CSCs. EP2 and EP4 protein was detected for MCF-7, MDA-MB-231, and SUM149 CSCs. The Celecoxib IC50 values for MCF-7 and SUM149 CSC spheroid formation were 0.5 and 2.2 uM, respectively; GW627386X IC50 values for MCF-7 and SUM149 CSC spheroid formation were 1.2 and 0.3uM, respectively. No significant differences in PGE2 production were observed for MCF-7 CSCs compared to adherent MCF-7 cells (11.5 ± 4.4 vs. 8.7 ± 2.1 pg/mL/106 cells; p=0.88). However, PgE2 production in MDA-MB-231 CSCs was significantly higher than adherent MDA-MB-231 cells (1507.0 ± 329.0 vs. 13.8 ± 5.7 pg/mL/106 cells; p≤0.001) and SUM149 CSC PgE2 production was significantly higher than SUM149 adherent cells (4932.9 ± 501.7 vs. 194.5 ± 31.0 pg/mL/106 cells; p≤0.001). 10uM Celecoxib treatment inhibited PGE2 production in MDA-MB-231 and MDA-MB-231 CSCs. Conclusions: MDA-MB-231 and SUM149 CSCs express COX-2, EP2 and Ep4 and produce high levels of PGE2. CSC spheroid formation is significantly decreased with COX-2 and EP4 inhibition.

Multiple‐staged Regulation of Myogenic Differentiation by Prostaglandin E2

We showed that conditioned media from osteocytes accelerates myogenesis in C2C12 myoblasts, which suggests signaling from bone to muscle cells. Since osteocytes secrete Prostaglandin E2 (PGE2), one of the major PGs synthesized from arachidonic acid through cyclooxygenase pathways, we started a systematic investigation of PGE2 roles in myogenesis. C2C12 myoblasts were used to investigate the effect and related molecular mechanisms of PGE2 on myogenic differentiation. Expression of PGE2 receptors (EP1‐EP4) was determined by real‐time PCR. EP1 and EP4 were highly expressed, whereas EP2 and EP3 were not detectable. Specific inhibitors SC 51322 for EP1 or L161,982 for EP4 significantly attenuated myogenesis. EP1 inhibition reduced the number of early myotubes, and subsequently affected the development of mature myotubes. On the other hand, EP4 inhibition did not affect the generation of early myotubes, but the fusion of early myotubes was dramatically inhibited. Interestingly, expression of two key myogenic regulatory factors, MyoD and Myogenin, was downregulated by EP1, but not by EP4 inhibition. These data provide evidence for the regulation of myogenesis by PGE2 via EP1 and EP4. The different effects of EP1 and EP4 inhibition suggest PGE2 signaling in myogenic differentiation is complex and multiple‐faceted. Support: NIH RC2 AR058962, Missouri Life Sciences Research Board(MB), and NIDCR DE021888(OJI).

Abstract 4007: Defining the role of ERG-mediated repression of HPGD in prostate tumorigenesis

Abstract Activation of the ETS Related Gene (ERG) was originally identified in subsets of Ewing sarcomas, myeloid leukemias and, recently, in the majority of prostate cancers (CaP). Emerging studies in experimental models underscore functions of ERG in prostate tumorigenesis. However, biological functions of ERG in CaP remains to be better understood. Evaluation of ERG downstream targets identified 15-hydroxyprostaglandin dehydrogenase gene (HPGD) in response to ERG knockdown. Several recent studies have indicated that HPGD, the main catabolizing enzyme of prostaglandins and lipoxins and a tumor suppressor, is down-regulated in a majority of lung, colon, breast, and bladder cancers. However, the regulation of HPGD within the biological context of ERG expressing prostate tumors remains to be defined. The objective of this study was to evaluate the effects of ERG on the prostaglandin signaling pathway in prostate cancer. Correlation between HPGD and ERG expression was evaluated at the mRNA and protein levels in prostate cancer specimens and cell lines by Western blot, QRT-PCR, and immunofluorescence assays. Small interference RNA against human ERG and HPGD were used to knock-down their respective expressions. Functional consequences of ERG and HPGD knockdown in VCaP cells harboring TMPRSS2-ERG fusions were assessed by ChIP and BrdU cell proliferation assays. The experiments showed that loss of ERG expression leads to a robust overexpression of HPGD in prostate cancer cells. Furthermore, we observed altered PGE2 production, decreased expression of prostaglandin E2 receptor 4 (EP4), and inhibition of prostaglandin E2 induced cell growth. Chromatin immunoprecipitation experiments revealed direct binding of ERG protein to the HPGD core promoter. Comparison of HPGD expression in TMPRSS2-ERG positive and negative prostate tumors indicated a trend towards decreased HPGD expression. In conclusion ERG activation, as a consequence of TMPRSS2-ERG fusions, may influence the components of the prostaglandin signaling pathway in prostate tumorigenesis disrupting the tumor suppressor functions of HPGD. These findings suggests for a new biological role of ERG in CaP that may be further evaluated as a target for prevention, as well as early therapeutic intervention in CaP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4007. doi:10.1158/1538-7445.AM2011-4007

Hormonal Regulation of Prostaglandin E2 Receptors: Localization and Expression in Rat Cervical Tissue

Prostaglandin E2 (PGE2) may regulate uterine activation and cervical ripening for labor through specific contractile and relaxatory receptors (EP1-4). The aim of this study was to determine the expression of PGE2 receptor isoforms in pregnant rat cervix during RU486-induced labor and progesterone supplementation to delay labor. Localization and expression of cervical PGE2 receptors were evaluated, and quantitative real-time polymerase chain reaction (PCR) for EP1-4 was performed. EP1-4 were found in both cervical epithelium and smooth muscle. RU486 treatment increased EP2 and EP4 messenger RNA (mRNA) and protein expression. Progesterone treatment had no effect on EP2 and EP4 mRNA expression but decreased EP4 protein. Hormonal manipulation resulted in differences in cellular localization of EP1 and EP3 in cervical epithelial cells, suggesting a specific role in that cell. Progesterone differentially regulates the expression of PGE2 receptor isoforms in the cervix. Elucidating the regulation of PGE2 receptors may facilitate improved approaches to the prevention and treatment of preterm labor.

Spatial and Temporal Expression of Prostaglandin E2 Receptors in Uterus and Conceptus During the Estrous Cycle and Early Pregnancy in Sheep.

Spatial and Temporal Expression of Prostaglandin E2 Receptors in Uterus and Conceptus During the Estrous Cycle and Early Pregnancy in Sheep.

Expression of prostaglandin E2 receptors in oral squamous cell carcinomas and growth inhibitory effects of an EP3 selective antagonist, ONO-AE3-240

Prostaglandin E2 (PGE2) can stimulate tumor progression by both direct and indirect mechanisms. However, its influence on cell proliferation is still unclear. The present study characterized expression of subtypes of PGE2 receptors in oral squamous cell carcinomas, while also investigating the effects of EP3 and EP4 selective antagonists on oral carcinoma cell lines. EP1, EP2, EP3 and EP4 receptor mRNAs were detected in 4, 5, 10 and 10 of 11 surgical specimens respectively. Application of an EP3 antagonist (ONO-AE3-240) strongly inhibited cell growth in COX-2 and PGE2 high expression cells (Ca9-22) but not in COX-2 and PGE2 low expression cells (HSC4). The antagonist also reduced the production of endogenous PGE2 and induced G0/G1 phase cell arrest. Addition of exogenous PGE2 only partly abrogated the growth inhibition, indicating that the anti-proliferative effect via EP3 receptor signaling was not only due to PGE2-dependent but also PGE2-independent mechanisms. In contrast, an EP4 antagonist (ONO-AE3-208) did not inhibit growth in either of the cancer cell lines. In summary, PGE2 receptor EP3 signaling probably contributes to development of oral carcinomas and use of EP3 antagonist may be a new therapeutic strategy for head and neck cancer.