EP2 and EP3 receptors as therapeutic targets for underactive bladder/detrusor underactivity due to diabetic cystopathy in a type 1 diabetic rat model.

OBJECTIVES: Diabetic cystopathy (DC) is recognized as one of the major etiologies of underactive bladder (UAB)/detrusor underactivity (DU). Although DC was first reported about three decades ago, there is a distinct lack of effective pharmacological management methods for UAB/DU due to DC with a robust certainty of evidence. In this study, we investigated whether EP2 and EP3 receptors are promising targets of pharmacological management of UAB/DU due to DC. METHODS: We used streptozotocin (STZ)-induced diabetic Sprague-Dawley rats with postvoid residual urine (PVR) greater than 0.1 mL. Sixteen weeks after induction of diabetes, we performed awake single cystometry after oral administration of the vehicle, an α-blocker (tamsulosin [TAM], 0.1 and 0.3 mg/kg), a cholinesterase inhibitor (distigmine [DIS], 0.3 and 1.0 mg/kg), or an EP2/3 dual agonist (ONO-8055, 0.01 and 0.03 mg/kg). We compared cystometric parameters after administration of the vehicle and drugs using a paired t test. P < .05 was considered to be statistically significant. RESULTS: Compared with the vehicle, TAM significantly decreased maximum intravesical pressure during voiding (Pmax), while DIS significantly increased it. However, neither drug significantly affected PVR or the residual urine rate (RUR). On the other hand, ONO-8055 significantly decreased PVR and tended to decrease RUR, although it did not significantly affect Pmax. CONCLUSION: The present study was unable to demonstrate that stimulation of EP2 and EP3 receptors caused major improvements in UAB/DU due to DC. However, this equivocal result could arise from inherent limitations of the STZ-induced diabetic rat as a UAB/DU model.

COX-2/EP2-EP4/ß-catenin signaling regulates patulin-induced intestinal cell proliferation and inflammation.

Patulin (PAT), a mycotoxin, is a natural contaminant that is produced by certain species of Penicillium, Aspergillus and Byssochlamys. The major contamination of PAT is in apple and apple based products. PAT is known to cause glutathione depletion, oxidative DNA damage and cell proliferation. Recently, in vitro studies have indicated that PAT can also increase the intestinal epithelial permeability, modulate tight junctions and decrease transepithelial electrical resistance. Nonetheless, no previous study has evaluated the mechanisms responsible for PAT-induced intestinal toxicity or its relevance to the in vivo situation. Here, Wistar rats were orally treated with 100 µg/kg body weight (b.wt.) of PAT, either alone or along with 100 mg/kg b. wt. of celecoxib for 3 days. We found that PAT exposure led to significantly higher levels of PGE2 in serum and intestinal tissue and high expression of COX-2 and Ki-67 compared to controls. Interestingly, our results showed that celecoxib treatment could decrease the PAT-induced PGE2 and reduce the PAT-induced intestinal damage. To study the mechanistic aspect, normal rat intestinal epithelial cells (IEC-6) were treated with non-toxic concentrations (100 nM, 250 nM and 500 nM) of PAT for 6 h. It was observed that PAT exposure caused enhanced proliferation, higher expression of COX-2, and EP2 and EP4 receptors, along with increased PGE2 secretion. Additionally, PAT exposure caused enhanced Akt expression, which in turn inhibits GSK-3ß and stabilizes ß-catenin. Overall, our study suggests that the COX-2/EP2-EP4/ß-catenin signaling cascades are involved in the regulation of PAT-induced intestinal cell proliferation and inflammation.

Discovery of 2-Piperidinyl Phenyl Benzamides and Trisubstituted Pyrimidines as Positive Allosteric Modulators of the Prostaglandin Receptor EP2.

Prostaglandin E2 (PGE2) via its Gαs-coupled EP2 receptor protects cerebral cortical neurons from excitotoxic and anoxic injury, though EP2 receptor activation can also cause secondary neurotoxicity in chronic inflammation. We performed a high-throughput screen of a library of 292 000 small molecules and identified several compounds that have a 2-piperidinyl phenyl benzamide or trisubstituted pyrimidine core as positive modulators for human EP2 receptor. The most active compounds increased the potency of PGE2 on EP2 receptor 4-5-fold at 20 µM without altering efficacy, indicative of an allosteric mechanism. These compounds did not augment the activity of the other Gαs-coupled PGE2 receptor subtype EP4 and showed neuroprotection against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. These newly developed compounds represent second-generation allosteric potentiators for EP2 receptor and shed light on a promising neuroprotective strategy. They should prove valuable as molecular tools to achieve a better understanding of the dichotomous action of brain EP2 receptor activation.

Direct growth-inhibitory effects of prostaglandin E2 in pancreatic cancer cells in vitro through an EP4/PKA-mediated mechanism.

BACKGROUND: There is strong evidence linking inflammation and the development of pancreatic ductal adenocarcinoma. Cyclooxygenase-2 (COX-2) and COX-2-derived PGE2 are overexpressed in human and murine pancreatic ductal adenocarcinoma. Several studies have demonstrated an important role of COX-2-derived PGE2 in tumor-stroma interactions; however, the direct growth effects of prostaglandin E2 (PGE2) on pancreatic ductal adenocarcinoma cells is less well defined. Our aim was to investigate the effects of PGE2 on pancreatic ductal adenocarcinoma cell growth and to characterize the underlying mechanisms. METHODS: Human pancreatic ductal adenocarcinoma cell lines, Panc-1 and MIA PaCa-2, were treated with PGE2 in varying doses (0-10 µM). Effects on the phosphorylation of ERK1/2 were evaluated by Western blot. Colony formation was observed for cells treated with PGE2 for 11 days. DNA synthesis was determined by (3H)-thymidine incorporation assay. Gene expression of E-type prostaglandin (EP)2/EP4 receptors and their correlation with survival in patients with pancreatic ductal adenocarcinoma were assessed using the RNA-Seq data set from The Cancer Genome Atlas Research Network. RESULTS: PGE2 decreased the size and number of colonies in Panc-1 but not MIA PaCa-2 cells. In the Panc-1 cells, PGE2 activated PKA/CREB and decreased phosphorylation of ERK1/2, which was reversed by an EP4 receptor antagonist, while an EP2 receptor antagonist had no effect. In contrast, in MIA PaCa-2 cells, PGE2 had no effect on ERK1/2 phosphorylation. Treatment of both Panc-1 and MIA PaCa-2 cells with forskolin/IBMX decreased ERK1/2 phosphorylation. Finally, PGE2 decreased DNA synthesis only in Panc-1 cells, which was reversed by an EP4 receptor antagonist. In human pancreatic ductal adenocarcinoma, high EP2 and low EP4 gene expression was correlated to worse median overall survival (15.6 vs 20.8 months, log-rank P = .017). CONCLUSION: Our study provides evidence that PGE2 can inhibit directly pancreatic ductal adenocarcinoma cell growth through an EP4-mediated mechanism. Together with our gene expression and survival analysis, this observation suggests a protective role of EP4 receptors in human pancreatic ductal adenocarcinoma that expresses E-type prostaglandin receptors.

Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells.

PURPOSE: Prostaglandin F2α analogues are the first-line medication for the treatment of ocular hypertension (OHT), and prostanoid EP2 receptor agonists are under clinical development for this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) cells, which could be a causal mechanism of TM dysfunction in glaucoma. METHODS: Human primary TM cells were treated with either latanoprost or butaprost and TGF-ß2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cell-populated collagen gel (CPCG) model. Expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of actin stress fibers and collagen deposition were evaluated by immunocytochemistry. Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific inhibitors. RESULTS: In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, α-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-ß2-induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-ß2-dependent collagen deposition. The latanoprost effects were mediated by p38 pathway. CONCLUSIONS: Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-ß2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT.

Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.

Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1ß production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1ß/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1ß/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1ß inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.

Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

Vasodilatory effect of 14,15-epoxyeicosatrienoic acid on mesenteric arteries in hypertensive and aged rats.

The objective of this study was to investigate the 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced vasodilatations as well as the underlying signaling pathways in rat mesenteric arteries from young, adult and old normotensive (WKY) and hypertensive rats. Protein expressions for prostaglandin EP(1-4) receptors, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, and adenylate cyclase (AC) were determined together with 14,15-EET-induced vasodilatations in primary- versus secondary-branches of the mesenteric artery. Responses to 14,15-EET were greater in the smaller secondary- versus primary-branches (and also more sensitive with lower EC50) and were reduced in vessels from old (80 weeks) rats as well as from vessels from the spontaneous hypertensive rats (SHR). Regardless of age or hypertension responses to 14,15-EET were inhibited by the EP2 antagonist AH6809, BK(Ca) channel inhibitor iberiotoxin, or 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway antagonists. These data indicate 14,15-EET-induced vasodilatation is mediated via the activation of EP2 receptors and opening of BK(Ca) channels. The expressions of the EP2 receptor and AC were markedly reduced in vessels from SHR as well as old rats, whereas BK(Ca) expression was reduced in old WKY and SHR, but not adult SHR. Furthermore, expression of the p53 protein, an indicator of cell senescence and apoptosis, was elevated in adult and old SHR as well as in old WKY. In summary, attenuated 14,15-EET-induced vasodilatation in mesenteric arteries from old normotensive WKY as well as adult and old SHR is associated with reduced expression of EP2 receptors and AC.

Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2.

BACKGROUND AND OBJECTIVE: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. MATERIAL AND METHODS: HPDL cells were cultured with 0.001-1 µm PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. RESULTS: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dose-dependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. CONCLUSION: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.

Prostaglandin receptor EP2 in the crosshairs of anti-inflammation, anti-cancer, and neuroprotection.

Modulation of a specific prostanoid synthase or receptor provides therapeutic alternatives to nonsteroidal anti-inflammatory drugs (NSAIDs) for treating pathological conditions governed by cyclooxygenase-2 (COX-2 or PTGS2). Among the COX-2 downstream signaling pathways, the prostaglandin E2 (PGE2) receptor EP2 subtype (PTGER2) is emerging as a crucial mediator of many physiological and pathological events. Genetic ablation strategies and recent advances in chemical biology provide tools for a better understanding of EP2 signaling. In the brain, the EP2 receptor modulates some beneficial effects, including neuroprotection, in acute models of excitotoxicity, neuroplasticity, and spatial learning via cAMP-PKA signaling. Conversely, EP2 activation accentuates chronic inflammation mainly through the cAMP-Epac pathway, likely contributing to delayed neurotoxicity. EP2 receptor activation also engages ß-arrestin in a G-protein-independent pathway that promotes tumor cell growth and migration. Understanding the conditions under which multiple EP2 signaling pathways are engaged might suggest novel therapeutic strategies to target this key inflammatory prostaglandin receptor.

Novel prostaglandin receptor modulators--part II: EP receptor modulators; a patent review (2002 - 2012).

INTRODUCTION: Prostaglandins and their G-protein-coupled receptors play numerous physiological and pathophysiological roles, especially in inflammation and its resolution. The variety of effects mediated by prostanoids makes prostanoid receptors valuable drug targets and the research on prostaglandin receptor modulators is intensive. The physiological and pathophysiological effects of prostaglandin E(2) are especially complex and numerous. The four subtypes of EP receptor have gained a lot of industrial and academic interest, in particular EP(2) and EP(4) for various indications. AREAS COVERED: Evaluation of the patent activity over the last decade (2002 - 2012) illustrates several potent compounds targeting the distinct prostaglandin E(2) receptors. Many novel methods for the use of EP receptor modulators have been developed, in addition to the classical indications for agents modulating the arachidonic acid cascade such as pain and inflammation. EXPERT OPINION: Several EP targeting agents with good potency and selectivity have been developed but their pharmacological use and utility has not yet been satisfactorily investigated. More research is necessary, and clinical use of these agents might therefore take some more time.

Distinct roles of central and peripheral prostaglandin E2 and EP subtypes in blood pressure regulation.

Prostaglandin E(2) (PGE(2)) is a major prostanoid with a wide variety of biological activities. PGE(2) can influence blood pressure (BP) both positively and negatively. In particular, centrally administered PGE(2) induces hypertension whereas systemic administration of PGE(2) produces a hypotensive effect. These physiologically opposing effects are generated by the existence of multiple EP receptors, namely EP(1-4), which are G protein-coupled receptors with distinct signaling properties. This review highlights the distinct roles of PGE(2) in BP regulation and the involvement of specific EP receptor subtypes.

Potential therapeutic interventions via EP2/EP4 prostaglandin receptors.

Prevention and treatment of pathological inflammatory processes requires application of various classes of immune suppressors, such as calcineurin inhibitors, steroids and non-steroid inhibitors of prostaglandin synthesis. However, each type of these immune suppressors causes less or more serious adverse side-effects. Exploration of the role played by prostanoids in the immune response and identification of functionally distinct prostaglandin E receptors (EP1-EP4) opened new perspectives in therapy of inflammation, autoimmunity and prevention of graft rejection. The EP4 receptor appeared to be an attractive target to affect manifestations of various pathological states by application of either agonists or antagonists of the receptor. This article presents a short overview of experimental approaches aimed at manipulation of signaling via EP2 and EP4 receptors that could have therapeutic utility.

Prostaglandin E2 represses interleukin 1 beta-induced inflammatory mediator output from pregnant human myometrial cells through the EP2 and EP4 receptors.

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals.

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.

Towards developing new strategies to reduce the adverse side-effects of nonsteroidal anti-inflammatory drugs.

The antipyretic and analgesic actions of nonsteroidal anti-inflammatory drugs (NSAIDs) are caused by the inhibition of prostaglandin E(2) (PGE(2)), thromboxane A(2) and prostacyclin (PGI(2)) production. Accumulating evidence suggests that the inhibition of PGE(2) production can cause adverse side-effects of NSAIDs on fluid and blood pressure regulation, such as hypertension and edema formation. Since both cyclooxygenase (COX)-1 and COX-2 isoforms contribute to the production of PGE(2), selective COX-2 inhibitors are not free of these adverse side-effects although they may be less severe. Four subtypes of PGE(2) receptors have been identified. The antipyretic action of blunted PGE(2) production is mediated predominantly by a reduced input to the prostaglandin E receptor 3 (EP(3)) pathway, whereas the analgesic action is mediated predominantly by a reduced input to the EP(1) pathway and perhaps by contributions from the other EP receptors. Accordingly, some of the adverse side-effects might be moderated by combined use of NSAIDs with selective EP(2) or EP(4) agonists that do not block the antipyretic or analgesic actions of NSAIDs that are mediated by reduced activation of EP(1) or EP(3) receptors. Moreover, EP(2) receptor-deficient mice had salt-sensitive hypertension and EP(4) receptor blockade moderated salt and water excretion and both EP(2) and EP(4) agonists had renoprotective effects. This suggests that strategies to maintain activation of EP(2) and EP(4) receptors during NSAID administration may not only reduce adverse effects but might confer additional benefits. In conclusion, enhancing EP(2) and EP(4) receptor activity by administration of selective agonists during the administration of NSAIDs has the potential to permit treating fever, inflammation and pain but with marginal adverse effects on fluid or blood pressure regulation.

Sodium butyrate induces the production of cyclooxygenases and prostaglandin E2 in ROS 17/2.8 osteoblastic cells.

OBJECTIVE: Sodium butyrate (butyric acid; BA) is a major metabolic by-product of the anaerobic periodontopathic bacteria present in subgingival plaque. We examined the effects of BA and/or indomethacin on cell proliferation, the expression of cyclooxygenases (COXs), prostaglandin (PG) receptors (EP1-4), extracellular matrix proteins, such as type I collagen and osteopontin, and PGE(2) production, using ROS17/2.8 cells as osteoblasts. METHODS: The rat clonal cell line ROS 17/2.8 was cultured with 0, 10(-5), 10(-4), and 10(-3)M BA in the presence or absence of 0.5 µM indomethacin, for up to 7 days. The expression of COX-1, COX-2, EP1, EP2, EP3, EP4, type I collagen, and osteopontin was examined at the mRNA and protein levels using real-time PCR and Western blotting, respectively. The amount of PGE(2) in the culture medium was measured by ELISA. RESULTS: Proliferation of ROS 17/2.8 cells was not affected by the addition of BA. However, PGE(2) production and the expression of COX-1 and COX-2 increased with the addition of BA. In contrast, indomethacin, an inhibitor of COX, blocked the stimulatory effect of BA. Furthermore, EP2 expression increased with BA treatment, whereas EP1 expression was not affected and the expression of EP3 and EP4 was not detected. The addition of BA also increased the expression of type I collagen and osteopontin. Indomethacin blocked about 50% of the stimulatory effect of BA on type I collagen, whereas it did not block the effect on osteopontin. CONCLUSIONS: These results suggest that BA induces PGE(2) production by increasing the expression of COX-1 and COX-2 in osteoblasts, and that an autocrine action of the produced PGE(2), via EP1 or BA-induced EP2, is related to an increase in type I collagen expression by BA.