PGE2-mediated Signaling Research Articles

Regulatory effects of paeoniflorin-6'-O-benzene sulfonate (CP-25) on dendritic cells maturation and activation via PGE2-EP4 signaling in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. Dendritic cells (DCs) are one of the most powerful antigen-presenting cells, and they play an important role in RA pathogenesis. Prostaglandin E2 (PGE2) is a potent lipid mediator that can regulate the maturation and activation of DCs, but the molecular mechanisms have not been elucidated. In this study, both in vitro and in an RA rat model, we investigated the mechanisms involved by focusing on PGE2-mediated signaling and using a novel anti-inflammatory compound, paeoniflorin-6'-O-benzene sulfonate (CP-25). PGE2 combined with tumor necrosis factor-α promoted DC maturation and activation through EP4-cAMP signaling. Treatment with CP-25 increased the endocytic capacity of DCs induced by PGE2. CP-25 inhibited the potency of DCs induced by the EP4 receptor agonist, CAY10598, to stimulate allogeneic T cells. Consistent with these findings, the CAY10598-induced upregulation of DC surface activation markers and production of IL-23 was significantly inhibited by CP-25 in a concentration-dependent manner. In vivo administration of CP-25 alleviated adjuvant arthritis (AA) in rats through inhibition of DC maturation and activation. Our results indicate that PGE2-EP4-cAMP signal hyperfunction can lead to abnormal activation of DC functions, which correlates with the course of disease in AA rats and provides a possible treatment target. The inhibition of DC maturation and activation by CP-25 interference of the PGE2-EP4 pathway may significantly contribute to the immunoregulatory profile of CP-25 when used to treat RA and other immune cell-mediated disorders.

COX-1/PGE2/EP4 alleviates mucosal injury by upregulating \u03b2-arr1-mediated Akt signaling in colitis

COX-1/PGE2 is an important protective mediator in ulcerative colitis (UC). β-arrestin1 (β-arr1), which acts as a scaffold protein, is involved in PGE2-mediated signaling pathways. However, the interaction between PGE2 and β-arr1 in maintaining mucosal barrier integrity remains unexplored. In this study, we demonstrated that COX-1 and PGE2 were significantly decreased, and EP4 mRNA was downregulated in both UC patients and mice during the injury phase. PGE2 treatment was found to alleviate mucosal injury and induce EP4 expression during dextran sulfate sodium (DSS)-induced colitis in wild-type (WT) mice. Following DSS-induced injury, β-arr1 deficient mice showed increased signs of colitis compared to β-arr1 WT mice, and the expression of PI3K and p-Akt were remarkably downregulated in β-arr1 deficient mice. In parallel, HCT116 cells transfected with β-arr1 siRNA were examined in the presence or absence of PGE2in vitro. PGE2 treatment in the β-arr1 WT/KO DSS model and β-arr1 siRNA transfection of HCT116 cells confirmed that PGE2 upregulated β-arr1 in vivo and in vitro. Collectively, our results indicate that COX-1/PGE2/EP4 upregulates the β-arr1 mediated Akt signaling pathway to provide mucosal protection in colitis. Thus, these findings provide support for the future development and clinical application of COX-1/PGE2 in UC.

Abstract P3-12-06: Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel

Abstract Paclitaxel (PTX), a microtubule-targeting anticancer agent, produces a debilitating peripheral neuropathy that is accompanied by neuropathic pain. Patients develop localized pain in their distal extremities, and more frequently complain of increased sensitivity to cold rather than hot temperatures. These clinical observations led us to hypothesize that PTX differentially damages subpopulations of peripheral sensory neurons. The purpose of this study was to evaluate the effects of PTX on bradykinin (BK) and eicosanoid (EP) receptor-expressing sensory neurons. PTX (11.7 mg/kg) or vehicle (Cremophor EL/EtOH/PBS; 1:1:6) was administered intraperitoneally to Sprague-Dawley rats (250-300 g) every other day for 5 days. Following treatment, PTX-treated rats exhibited increased sensitivity to cold and mechanical stimuli, but decreased sensitivity to heat stimulation as compared to vehicle-treated rats. Intraplantar (i.pl.) injection with BK (5 μg) produced a prolonged allodynia to cold, but a diminished allodynia to heat in PTX-treated rats. Interestingly, following treatment with PGE2 (0.3 μg, i.pl.), PTX-treated rats displayed no statistically significant differences in the allodynia to cold or heat compared to vehicle-treated rats. Efficacy of BK (1 pM - 1 μM) to stimulate inositol phosphate (IP) production in cultured primary sensory neurons from PTX-treated rats was significantly reduced. By contrast, stimulation of cAMP production by PGE2 (0.01 pM - 100 nM) was unaffected by PTX treatment. Preliminary mechanistic studies treating naïve cultures of rat primary sensory neurons directly with PTX (5 nM, 0 - 48 hr) yielded a time-dependent, tri-phasic effect on BK-stimulated IP accumulation and reduced PGE2-stimulated cAMP production, suggesting PTX differentially disrupts BK- and PGE2-mediated signaling. Collectively, these data provide evidence that subpopulations of peripheral sensory neurons are differentially sensitive to PTX. Understanding why particular subclasses are less sensitive, or resistant, to damage could reveal novel approaches to protect sensory neurons from chemotoxicity and ultimately prevent chemotherapy-induced peripheral neuropathy. Supported by USPS grant #8ULITR000149, Greehey Fellowship, and Translational Science Training Across Disciplines Program at UTHSCSA The project described was supported by Award Number 8UL1TR000149 from the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Advancing Translational Sciences. Citation Format: Peter M LoCoco, Teresa C Chavera, Raehannah J Jamshidi, Susan L Mooberry, Kelly A Berg, William P Clarke. Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-12-06.

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Modulation of Host Natural Killer Cell Functions in Breast Cancer via Prostaglandin E2 Receptors EP2 and EP4

Breast malignancies often have high levels of COX-2. The COX-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor EP4 (1 of 4 EP expressed on the malignant cell) reduces metastasis by a mechanism that requires natural killer (NK) cells. Tumor-derived PGE2 and exogenous PGE2 are known to have direct inhibitory effects on NK cell functions, but less is known regarding which EP receptors mediate these effects. We now show that several NK functions (lysis, migration, cytokine production) are compromised in tumor-bearing mice and that tumor-produced PGE2 interferes with NK cell functions. PGE2 inhibits the potential of NK cells to migrate, exert cytotoxic effects, and secrete interferon γ. The ability of PGE2 to inhibit NK cells from tumor-bearing mice is by acting on EP2 and EP4 receptors. NK cells from tumor-bearing mice were more sensitive to inhibition by EP4 and EP2 agonists compared with endogenous NK cells from healthy mice. PGE2 was inhibitory to most NK functions of either normal or tumor-bearing mice. In contrast, there was a trend for enhanced tumor necrosis factor α production in response to PGE2 by NK cells from tumor-bearing mice. We also report that a recently described EP4 antagonist, frondoside A, inhibits breast tumor metastasis in an NK-dependent manner and protects interferon γ production by NK cells from PGE2-mediated suppression. Taken together these data show that NK functions are depressed in tumor-bearing hosts relative to normal NK cells and that PGE2 suppresses NK functions by acting on EP2 and EP4 receptors.

EP Receptor Expression in Human Intestinal Epithelium and Localization Relative to the Stem Cell Zone of the Crypts

There is substantial evidence for PGE2 affecting intestinal epithelial proliferation. PGE2 is also reported to be involved in the regulation of growth and differentiation in adult stem cells, both effects mediated by binding to EP-receptors. We have used the Lgr5 as a marker to scrutinize EP-receptor and COX expression in human intestinal epithelial cells with focus on the stem cell area of the crypts. Normal tissue from ileum and colon, but also duodenal biopsies from patients with untreated celiac disease, were investigated by immunohistochemistry and RT-PCR. The combination of fresh flash-frozen tissue and laser microdissection made it possible to isolate RNA from the epithelial cell layer, only. In the small intestine, Lgr5 labels cells are in the +4 position, while in the colon, Lgr5 positive cells are localized to the crypt bottoms. Epithelial crypt cells of normal small intestine expressed neither EP-receptor mRNA nor COX1/2. However, crypt cells in tissue from patients with untreated celiac disease expressed EP2/4 receptor and COX1 mRNA. In the colon, the situation was different. Epithelial crypt cells from normal colon were found to express EP2/4 receptor and COX1/2 transcripts. Thus, there are distinct differences between normal human small intestine and colon with regard to expression of EP2/4 receptors and COX1/2. In normal colon tissue, PGE2-mediated signaling through EP-receptors 2/4 could be involved in regulation of growth and differentiation of the epithelium, while the lack of EP-receptor expression in the small intestinal tissue exclude the possibility of a direct effect of PGE2 on the crypt epithelial cells.

Abstract 5308: Targeting the Cox2 pathway prostaglandin EP receptors to regulate NK cell functions

Abstract Breast malignancies often have high levels of Cox-2. The Cox-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicates that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor (EP4) reduces metastasis by a mechanism that requires Natural Killer (NK) cells. In order to better understand the mechanism by which inhibiting PGE2 production or signaling reduces metastatic disease, we examined the role of PGE2 in regulating protective host effector cells. It is known that NK cell function is compromised by PGE2, but very little is known about the mechanism by which PGE2 affects NK effector activity. NK effector function is regulated by complex interactions of recognition receptors expressed on NK cells and cognate ligands expressed on target cells. Both stimulatory and inhibitory signals are delivered through these receptor-ligand interactions. We have previously shown that both Cox inhibitors and antagonists of the EP4 receptor modulate the expression of NK cell stimulatory (H60) and inhibitory (MHC class I) ligands on the tumor target cell in favor of NK recognition and killing. We now report the direct effects of PGE2 on the NK cell. Murine splenic NK cells express all four PGE2 receptors (EP1-4). We examined the role of EP receptors in three NK cell functions; cytokine release, cytotoxicity, and migration. Activating all four NK-EP receptors with PGE2 leads to inhibited IFNγ secretion, induced TNFα production, less migration of NK cells and reduced ability to lyse tumor target cells. Using agonists and antagonists of individual EP receptors, we showed that only certain EP receptors contribute to loss of function. Next, we showed that all NK-EP receptor expression is downregulated during progressive tumor growth, which produced an environment that induced a less mature NK phenotype. EP2 and EP4 are linked to protein kinase A and elevations in intracellular cAMP. NK cells from tumor bearing mice respond poorly to stimulation with 10µM PGE2 and intracellular cAMP levels were increased by only 1.45-1.78 fold. In contrast, cAMP was induced by 6.08-10.58-fold in NK cells from normal mice. Thus, the tumor milieu results in changes in both NK-EP expression levels and EP receptor functions. NK mediated cytotoxicity, cytokine release, and migration were also compromised in tumor bearing mice. These results taken together support a mechanism whereby inhibiting PGE2 production or preventing signaling through some, but not all EP receptors may prevent suppression of NK functions that are critical to control breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5308.

EP1 Receptor-Mediated Migration of the First Trimester Human Extravillous Trophoblast: The Role of Intracellular Calcium and Calpain

The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta. Because EVT migrate into the prostaglandin (PG) E2-rich decidua, we tested the roles of PGE2 and PGE2-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture. mRNA expression of different PGE2 receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca2+ ([Ca2+]i) and calpain activity. Exogenous PGE2 stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1-4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca2+]i were confirmed by the following findings: 1) exogenous PGE2 or an EP1 agonist, but not an EP4 agonist, increased [Ca2+]i, which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE2-mediated migratory response of EVT; and 3) PGE2-mediated EVT migration was shown to be dependent on a class of Ca2+-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE2 stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration. PGE2 stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+]i, and activating calpain.

NaCl transport across the opercular epithelium ofFundulus heteroclitusis inhibited by an endothelin to NO, superoxide, and prostanoid signaling axis

Recent evidence suggests that paracrine signaling agents, such as endothelin (ET), nitric oxide (NO), superoxide (O2-), and prostanoids can modulate mammalian renal function by affecting both hemodynamic and epithelial ionic transport pathways. Since these signaling pathways have been described in fish blood vessels, we hypothesized that they may control salt transport across the gill epithelium--the primary site of ion excretion in marine teleost fishes. We found that ET, the NO donors sodium nitroprusside and spermine NONOate, and the prostanoid PGE2 each can produce a concentration-dependent reduction in the short circuit current (Isc) across the isolated opercular epithelium of the killifish (Fundulus heteroclitus), the generally accepted model for the marine teleost gill epithelium. Sarafotoxin S6c was equipotent to ET-1, suggesting that ETB receptors are involved. Incubation with NG-nitro-L-arginine methyl ester (L-NAME) or indomethacin reduced the effect of subsequent addition of SRXS6c by 17 and 89%, respectively, suggesting the presence of an ET to NO and PGE axis. The effects of l-NAME and indomethacin were not additive, but the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) reduced the effect of SRXS6c by 34% and preincubation with l-NAME, indomethacin, and TEMPOL reduced the SRXS6c response to zero. This suggests a direct role for O2- in this axis. COX-2 appears to be the major enzyme involved in this axis because the specific COX-2 inhibitor NS-398 was twice as effective as the COX-1 inhibitor SC560 in inhibiting the SRXS6c effect. The Isc was stimulated by the EP2 agonist butaprost and inhibited by the EP(1,3) agonist sulprostone, suggesting both stimulatory and inhibitory PGE receptors in this tissue. Carbaprostacyclin (PGI2 analog), thromboxane A2, PGF(2alpha), and PGD2 did not affect the Isc. Our data are the first to suggest the importance of an ET-stimulated and NO-, O2(-)-, and PGE2-mediated signaling axis that can modify active extrusion of NaCl across the killifish opercular epithelium and, by inference, the marine teleost gill epithelium.

CALCIUM SIGNALING RESTITUTION PREVENTS T-CELL PROLIFERATIVE SUPPRESSION BY PROSTAGLANDIN E2

Previous studies from our laboratory have implicated a role for Ca2+ in prostaglandin E2 (PGE2)-induced suppression in T-cell proliferation during sepsis. The present study further elucidated the mechanism of PGE2-induced suppression in T-cell proliferation. We assessed whether prevention of the suppression in Ca2+ mobilization in PGE2-treated T-cells would restore proliferation. Rat splenic T-cell Ca2+ mobilization and proliferation were measured after stimulation of cells with concanavalin A (Con A) employing Fura-2 spectroscopy and cellular [3H]thymidine uptake techniques, respectively. PGE2 and other agents that directly up-regulate the PGE2-mediated cell signaling events (e.g., cholera toxin and forskolin), substantially suppressed both Con A-induced proliferation (p < .01) and Ca2+ mobilization in T-cells (p < .01). When stimulated with Con A plus ionomycin, [Ca2+]i in PGE2 treated T-cells (395 +/- 21, nM) was not significantly different (p > .05) from that observed in Con A-stimulated T-cells without the PGE2 exposure (351 +/- 8.6). The stimulation of PGE2-treated T-cells with ionomycin and Con A also significantly (p < .025), if not completely, prevented the PGE2-induced suppression in T-cell proliferation. These results suggest that the cross-talk between the TCR- and PGE2-mediated signaling in T-cells negatively modulates the TCR signals at the Ca2+ mobilization step and/or earlier to it.