Inhibition of the Prostaglandin EP-1 Receptor in Periosteum Progenitor Cells Enhances Osteoblast Differentiation and Fracture Repair.

Fracture healing is a complex and integrated process that involves mesenchymal progenitor cell (MPC) recruitment, proliferation and differentiation that eventually results in bone regeneration. Prostaglandin E2 (PGE2) is an important regulator of bone metabolism and has an anabolic effect on fracture healing. Prior work from our laboratory showed EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation. We also showed that bone marrow MSCs from EP1-/- mice exhibit increased osteoblastic differentiation in vitro. In this study we investigate the changes in the periosteal derived MPCs (PDMPCs), which are crucial for fracture repair, upon EP1 deletion. EP1-/- PDMPCs exhibit increased numbers of total (CFU-F) and osteoblastic colonies (CFU-O) as well as enhanced osteoblastic and chondrogenic differentiation. Moreover, we tested the possible therapeutic application of a specific EP1 receptor antagonist to accelerate fracture repair. Our findings showed that EP1 antagonist administration to wild type mice in the early stages of repair similarly resulted in enhanced CFU-F, CFU-O, and osteoblast differentiation in PDMPCs and resulted in enhanced fracture callus formation at 10 days post fracture and increased bone volume and improved biomechanical healing of femur fractures at 21 days post fracture.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood. METHODS: Synovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3-4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1ß, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist. RESULTS: CGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists. CONCLUSIONS: PGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Antagonism of the prostaglandin E2 EP1 receptor in MDCK cells increases growth through activation of Akt and the epidermal growth factor receptor.

The actions of prostaglandin E2 (PGE2) in the kidney are mediated by G protein-coupled E-prostanoid (EP) receptors, which affect renal growth and function. This report examines the role of EP receptors in mediating the effects of PGE2 on Madin-Darby canine kidney (MDCK) cell growth. The results indicate that activation of Gs-coupled EP2 and EP4 by PGE2 results in increased growth, while EP1 activation is growth inhibitory. Indeed, two EP1 antagonists (ONO-8711 and SC51089) stimulate, rather than inhibit, MDCK cell growth, an effect that is lost following an EP1 knockdown. Similar observations were made with M1 collecting duct and rabbit kidney proximal tubule cells. ONO-8711 even stimulates growth in the absence of exogenous PGE2, an effect that is prevented by ibuprofen (indicating a dependence upon endogenous PGE2). The involvement of Akt was indicated by the observation that 1) ONO-8711 and SC51089 increase Akt phosphorylation, and 2) MK2206, an Akt inhibitor, prevents the increased growth caused by ONO-8711. The involvement of the EGF receptor (EGFR) was indicated by 1) the increased phosphorylation of the EGFR caused by SC51089 and 2) the loss of the growth-stimulatory effect of ONO-8711 and SC51089 caused by the EGFR kinase inhibitor AG1478. The growth-stimulatory effect of ONO-8711 was lost following an EGFR knockdown, and transduction of MDCK cells with a dominant negative EGFR. These results support the hypothesis that 1) signaling via the EP1 receptor involves Akt as well as the EGFR, and 2), EP1 receptor pharmacology may be employed to prevent the aberrant growth associated with a number of renal diseases.

COX-2 inhibitors arrest prostate cancer cell cycle progression by down-regulation of kinetochore/centromere proteins.

BACKGROUND: Previous studies have shown that COX-2 inhibitors inhibit cancer cell proliferation. However, the molecular mechanism remains elusive. METHODS: Prostate cancer LNCaP, 22Rv1, and PC3 cells were cultured and treated with the COX-2 inhibitors celecoxib and CAY10404. Knockdown of COX-2 in LNCaP cells was carried out using lentiviral vector-loaded COX-2 shRNA. Cell cycle progression and cell proliferation were analyzed by flow cytometry, microscopy, cell counting, and the MTT assay. The antagonists of EP1, EP2, EP3, and EP4 were used to examine the effects of the PGE2 signaling. The effect of COX-2 inhibitors and COX-2 knockdown on expression of the kinetochore/centromere genes and proteins was determined by RT-PCR and immunoblotting. RESULTS: Treatment with the COX-2 inhibitors celecoxib and CAY10404 or knockdown of COX-2 significantly inhibited prostate cancer cell proliferation. Flow-cytometric analysis and immunofluorescent staining confirmed the cell cycle arrested at the G2/M phase. Biochemical analysis showed that inhibition of COX-2 or suppression of COX-2 expression induced a dramatic down-regulation of key proteins in the kinetochore/centromere assembly, such as ZWINT, Cdc20, Ndc80, CENP-A, Bub1, and Plk1. Furthermore, the EP1 receptor antagonist SC51322, but not the EP2, EP3, and EP4 receptor antagonists, produced similar effects to the COX-2 inhibitors on cell proliferation and down-regulation of kinetochore/centromere proteins, suggesting that the effect of the COX-2 inhibition is through inactivation of the EP1 receptor signaling. CONCLUSIONS: Our studies indicate that inhibition of COX-2 can arrest prostate cancer cell cycle progression through inactivation of the EP1 receptor signaling and down-regulation of kinetochore/centromere proteins.

Prostaglandin E2 type 1 receptors contribute to neuronal apoptosis after transient forebrain ischemia.

Cyclooxygenase-2-derived prostaglandin E2 (PGE2) contributes to excitotoxic and ischemic neuronal cell death by engaging neuronal PGE2 type 1 receptors (EP1R). Our previous studies have shown that EP1R signaling resulted in disturbances of intracellular Ca(2+) homeostasis and suppression of the pro-survival protein kinase AKT. The aim of this study was to investigate whether these pathophysiological mechanism have a role in the neuronal cell death after transient forebrain ischemia. Mice were subjected to ischemia/reperfusion by bilateral common carotid artery occlusion. Hippocampal cornu ammonis area 1 (CA1) neuronal cell death was determined 5 days after reperfusion. Animals treated with the EP1R antagonist SC51089 or EP1R-deficient mice (EP1(-/-)) showed significantly less neuronal injury as compared to vehicle-treated wild-type controls. Benefits of EP1R blockage were still evident 14 days after injury. Better neuronal survival was correlated with reduced neuronal caspase-3 activity and decreased nuclear translocation of the apoptosis-inducing factor . Neuroprotection could be reverted by intracerebroventricular administration of the phosphoinositide 3-kinase inhibitor LY294002 and was not further increased by the calcineurin inhibitor FK506. These data implicate EP1R in postischemic neuronal apoptosis possibly by facilitating AKT inhibition.

Prostaglandin E2 receptor EP1 phosphorylate CREB and mediates MMP2 expression in human cholangiocarcinoma cells.

Cyclooxygenase-2 (COX-2) and COX-2-induced prostaglandin E2 (PGE2) have been implicated in all stages of malignant tumorigenesis. Although many aspects of matrix metalloproteinase (MMP2) on tumor invasion have been studied, the exact mechanism of PGE2-induced MMP2 overproduction has not been clearly defined. We have previously demonstrated that PGE2-enhanced extracellular signal-regulated kinase (Erk) phosphorylation via EP1 signaling pathway involved in PGE2-induced cell proliferation. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of MMP2 and Erk phosphorylate CREB at ser133, we hypothesize that CREB may be implicated in the signaling of PGE2 stimulation to MMP2 overproduction via EP1 receptor. In the study, we investigated the role of EP1 receptor on PGE2-induced MMP2 expression and delineated the signaling pathway that contributes to EP1 receptor modulation of MMP2 in human cholangiocarcinoma cells. We found PGE2 or selective EP1 receptor agonist 17-P-T-PGE2-stimulated MMP2 expression and selective EP1 receptor antagonist SC-51322 or EP1 receptor siRNA abrogated PGE2-induced MMP2 expression. Intracellular calcium chelator BAPTA-AM, the selective inhibitor of EGFR AG1478 and the selective inhibitor of Erk PD98059 blocked EP1 receptor activation-induced CREB phosphorylation and MMP2 expression. A novel dominant-negative (D-N) inhibitor protein of the CREB, termed A-CREB, attenuated EP1 receptor activation-induced MMP2 expression. Our findings suggest that PGE2-enhanced MMP2 expression is, at least in part, mediated through EP1 receptors and calcium signaling pathway-induced CREB phosphorylation in human cholangiocarcinoma cells.

The EP1 receptor for prostaglandin E2 promotes the development and progression of malignant murine skin tumors.

High levels of prostaglandin E2 (PGE2) synthesis resulting from the up-regulation of cyclooxygenase (COX)-2 has been shown to be critical for the development of non-melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G-protein coupled membrane receptors, EP1-4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCCs) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity, and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX-2 levels in the epidermis and SCC could be completely prevented in DMBA-treated BK5.EP1 mice either by feeding the selective COX-2 inhibitor celecoxib in their diet or by crossing them onto a COX-2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX-2.

The roles of prostaglandin EP 1 and 3 receptors in the control of human myometrial contractility.

CONTEXT: Prostaglandins are central to the processes of human labor. Prostaglandin E(2) (PGE(2)) synthesized within the uterus mediates cervical ripening and uterine contractions. PGE receptors, EP1 and EP3, may each mediate contractions, and represent potential therapeutic targets in the management of preterm labor. Studies of the expression and function of EP1 and EP3 in pregnant myometrium are inconsistent. OBJECTIVE: The objective of the study was to determine the relative importance of EP1 and EP3 in human myometrial contractility. DESIGN: We studied the expression of EP1 and EP3 in upper- and lower-segment myometrium at term in vivo and the effects of specific inhibitors on contractions in vitro. PATIENTS: Myometrial biopsies for both in vivo and in vitro studies were taken at cesarean section at term before or in labor in uncomplicated pregnancies. RESULTS: We found no differences in the expression of EP1 or EP3 at mRNA or protein level between the upper and lower segment myometrium and no overall changes associated with the onset of labor. Upon labor, EP1, but not EP3, was found to relocalize to the nucleus. In studies of contractility, we found no differences in spontaneous or PGE(2)-induced contractility between the upper- and lower-segment samples. Spontaneous contractions were inhibited by acetylsalicylic acid and were rescued by PGE(2). Although an EP1 antagonist, ZD6416, had no effect, an EP3 antagonist, L798106, inhibited both spontaneous and PGE(2)-induced contractions. CONCLUSIONS: EP3 is the primary receptor subtype that mediates PGE(2) induced contractility in human pregnant myometrium at term and represents a possible therapeutic target.

ANGPTL4 induction by prostaglandin E2 under hypoxic conditions promotes colorectal cancer progression.

Prostaglandin E(2) (PGE(2)), the most abundant COX-2-derived prostaglandin found in colorectal cancer, promotes tumor cell proliferation and survival via multiple signaling pathways. However, the role of PGE(2) in tumor hypoxia is not well understood. Here, we show a synergistic effect of PGE(2) and hypoxia on enhancing angiopoietin-like protein 4 (ANGPTL4) expression and that elevation of ANGPTL4 promotes colorectal cancer growth. PGE(2) induces ANGPTL4 expression at both the mRNA and protein levels under hypoxic conditions. Moreover, hypoxia induces one of the PGE(2) receptors, namely EP1. Activation of EP1 enhances ANGPTL4 expression, whereas blockage of EP1 by an antagonist inhibits PGE(2) induction of ANGPTL4 under hypoxic conditions. Importantly, overexpression of ANGPTL4 promotes cell proliferation and tumor growth in vitro and in vivo. In addition, treatment with ANGPTL4 recombinant protein increases colorectal carcinoma cell proliferation through effects on STAT1 signaling. The MAP kinase and Src pathways mediate ANGPTL4-induced STAT1 expression and activation. These results are relevant to human disease because we found that the expression of ANGPTL4 and STAT1 are elevated in 50% of human colorectal cancers tested and there is a positive correlation between COX-2 and ANGPTL4 as well STAT1 expression in colorectal carcinomas. Collectively, these findings suggest that PGE(2) plays an important role in promoting cancer cell proliferation via ANGPTL4 under hypoxic conditions.

EP1(-/-) mice have enhanced osteoblast differentiation and accelerated fracture repair.

As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro-computed tomographic (µCT), and biomechanical measures. EP1(-/-) mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1(-/-) mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1(-/-) mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE(2) receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.

Prostaglandin E receptor EP1 suppresses breast cancer metastasis and is linked to survival differences and cancer disparities.

Cyclooxygenase-2 is frequently overexpressed and associated with poor prognosis in breast cancer. The cyclooxygenase-2 product prostaglandin E(2) elicits cellular responses through four G-protein-coupled receptors, designated EP1 to EP4, coupled to distinct intracellular signaling pathways. EP4, expressed on malignant breast cells, promotes metastasis; however, a role for EP1 in metastasis has not been investigated. Using a murine model of metastatic breast cancer, we now show that pharmacologic antagonism of EP1 with SC19220 or AH6809 promoted lung colonization of mammary tumor cells by 3.7- to 5.4-fold. Likewise, reducing EP1 gene expression by shRNA also increased metastatic capacity relative to cells transfected with nonsilencing vector but did not affect the size of transplanted tumors. Examination of invasive ductal carcinomas by immunohistochemistry shows that EP1 was detected in both the cytoplasm and nucleus of benign ducts as well as malignant cells in some samples, but was absent or limited to either the nucleus or cytoplasm in other malignant samples. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression (P = 0.008). There was no difference in survival for women with differences in cytoplasmic EP1 expression (P = 0.46). Comparing EP1 mRNA in breast tumors from African American and European American women revealed that many more African American breast tumors lacked detectable EP1 mRNA (P = 0.04). These studies support the hypothesis that EP1 functions as a metastasis suppressor and that loss of nuclear EP1 is associated with poorer overall survival and may contribute to disparities in outcome in different populations.