Effect of deletion of the prostaglandin EP2 receptor on the anabolic response to prostaglandin E2 and a selective EP2 receptor agonist

Studies using prostaglandin E receptor (EP) agonists indicate that prostaglandin (PG) E(2) can have anabolic effects through both EP4 and EP2 receptors. We previously found that the anabolic response to a selective EP4 receptor agonist (EP4A, Ono Pharmaceutical) was substantially greater than to a selective EP2 receptor agonist (EP2A) in cultured murine calvarial osteoblastic cells. To further define the role of the EP2 receptor in PG-mediated effects on bone cells, we examined the effects of EP2A and PGE(2) on both calvarial primary osteoblasts (POB) and marrow stromal cells (MSC) cultured from mice with deletion of one (Het) or both (KO) alleles of the EP2 receptor compared to their wild-type (WT) littermates. Deletion of EP2 receptor was confirmed by quantitative real-time PCR, Western blot and immunohistochemistry. The 1 month-old mice used to provide cells in these studies did not show any significant differences in their femurs by static histomorphometry. EP2A was found to enhance osteoblastic differentiation as measured by alkaline phosphatase mRNA expression and activity as well as osteocalcin mRNA expression and mineralization in the WT cell cultures from both marrow and calvariae. The effects were somewhat diminished in cultures from Het mice and abrogated in cultures from KO mice. PGE(2) effects were greater than those of EP2A, particularly in POB cultures and were only moderately diminished in Het and KO cell cultures. We conclude that activation of the EP2 receptor is able to enhance differentiation of osteoblasts, that EP2A is a true selective agonist for this receptor and that PGE(2) has an additional anabolic effect likely mediated by the EP4 receptor.     

cAMP analogues downregulate the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in human bone marrow stromal cells in vitro.

The stimulation of granulocyte macrophage-colony stimulating factor (GM-CSF) by interleukin-1 (IL-1) has been shown to be counteracted in different mesenchymal cell systems by cyclic adenosine monophosphate (cAMP) agonists. The aim of this study was the evaluation of different cAMP agonists on GM-CSF expression in human bone marrow stromal cells. Incubation of secondary haematopoietic progenitor cell deprived human stromal cell cultures with IL-1 or TNF-alpha induced GM-CSF protein expression in culture supernatants and GM-CSF-mRNA in adherent stromal cells. The coincubation with 8-bromo-cAMP (8BrcAMP), a water soluble cAMP analogue, inhibited this GM-CSF stimulation at the protein and the mRNA level. This effect was dose dependent with a maximal inhibition of about 65% occurring at a 8BrcAMP concentration of 0.75 mM. In addition to 8BrcAMP, other cAMP agonists such as dibutyryl-cAMP, forskolin, pertussis toxin, or prostaglandin E2 (PGE2) had the same inhibitory effect on GM-CSF stimulation by IL-1. Coincubation with the cyclooxygenase inhibitor indomethacin had no significant influence on GM-CSF expression in stromal cells. Our results provide evidence that the previously described inhibitory effect of cAMP agonist PGE2 on haematopoietic progenitor cells in vivo is, at least in part, mediated by modulating the expression of GM-CSF in bone marrow stromal cells.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo

Prostaglandin E2 (PGE(2)), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4(flox/flox) mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE(2) or the EP4 selective agonists PGE(1)-OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4(flox/flox) cells, no effects were seen in adenoCre-transduced EP4(flox/flox) cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE(2)-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE(2), as well as PGE(1)-OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.     

Prostaglandin E(2) upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 macrophages

Prostaglandin E(2) (PGE(2)) has been implicated in the regulation of inflammatory and immunological events. Using RAW 264.7 macrophages, the present study investigates the influence of PGE(2) on the expression of cyclooxygenase-2 (COX-2). Incubation of cells with PGE(2) increased lipopolysaccharide (LPS)-induced COX-2 mRNA levels in a concentration-dependent manner. Upregulation of COX-2 expression by PGE(2) was completely abolished by the specific adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and mimicked by butaprost, a selective agonist of the adenylyl cyclase-coupled PGE(2) receptor subtype 2 (EP(2)), or 11-deoxy PGE(1), an EP(2)/EP(4) receptor agonist. By contrast, the EP(3)/EP(1) receptor agonists 17-phenyl-omega-trinor PGE(2) and sulprostone left LPS-induced COX-2 expression virtually unaltered. Upregulation of LPS-induced COX-2 expression and subsequent PGE(2) synthesis was also observed in the presence of the cell-permeable cAMP analogue dibutyryl cAMP and the adenylyl cyclase activator cholera toxin. Together, our data demonstrate that PGE(2) potentiates COX-2 mRNA expression via an adenylyl cyclase/cAMP-dependent pathway. In conclusion, upregulation of COX-2 expression via an autocrine feed-forward loop may in part contribute to the well-known capacity of PGE(2)/cAMP to modulate inflammatory processes.     

PGE2 enhances cytokine-elicited nitric oxide production in mouse cortical collecting duct cells.

It has been documented that arginine vasopressin (AVP) and prostaglandin E(2) (PGE(2)) regulate water reabsorption in renal tubular cells. The present study was attempted to delineate the downstream signaling of AVP and PGE(2) in a cortical collecting duct cell line (M-1 cell). Using RT-PCR, we detected mRNA for V2 and VACM-1 but not for V1a and AII/AVP receptors of AVP. Furthermore, neither AVP nor V2 receptor agonist and antagonist alter cellular cAMP. These together with unchanged cellular Ca(2+) by AVP suggested that AVP pathway was not operating in M-1 cells. All four classical PGE(2) receptors with EP3 and EP4 as the most prominent were detected in M-1 cells. PGE(2), 11-deoxy-PGE(1) (EP2 and EP4 agonist), and 17-phenyl-trinor-PGE(2) (EP1 agonist) increased cellular concentration of cAMP. There was no effect of PGE(2) or EP1 agonist on cellular Ca(2+). These findings provide evidence of the involvement of PGE(2) cascade in M-1 cells. M-1 cells were capable of synthesizing nitric oxide (NO). Although individual cytokines did not affect NO production, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma elevated NO concentration to 4.5-fold of the control. Addition of PGE(2) and db-cAMP to the cytokine mixture further increased NO production to 7.0- and 9.8-fold, respectively, of that seen in non-treated cells. PGE(2) or db-cAMP alone, however, had no effect on NO production. The results of the study led us to speculate that enhanced production of cAMP via PGE(2) signaling pathway in M-1 cells could either stimulate or attenuate water reabsorption in renal tubule. While an increase in cAMP alone may enhance water reabsorption, a concomitant increase in cAMP and cytokines may inhibit water reabsorption in renal tubule.     

Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.     

Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP

Prostaglandin E(2) is a potent lipid mediator of inflammation that effects changes in cell functions through ligation of four distinct G protein-coupled receptors (E-prostanoid (EP)1, EP2, EP3, and EP4). During pneumonia, PGE(2) production is enhanced. In the present study, we sought to assess the effect of endogenously produced and exogenously added PGE(2) on FcRgamma-mediated phagocytosis of bacterial pathogens by alveolar macrophages (AMs), which are critical participants in lung innate immunity. We also sought to characterize the EP receptor signaling pathways responsible for these effects. PGE(2) (1-1000 nM) dose-dependently suppressed the phagocytosis by rat AMs of IgG-opsonized erythrocytes, immune serum-opsonized Klebsiella pneumoniae, and IgG-opsonized Escherichia coli. Conversely, phagocytosis was stimulated by pretreatment with the cyclooxygenase inhibitor indomethacin. PGE(2) suppression of phagocytosis was associated with enhanced intracellular cAMP production. Experiments using both forskolin (adenylate cyclase activator) and rolipram (phosphodiesterase IV inhibitor) confirmed the inhibitory effect of cAMP stimulation. Immunoblot analysis of rat AMs identified expression of only EP2 and EP3 receptors. The selective EP2 agonist butaprost, but neither the EP1/EP3 agonist sulprostone nor the EP4-selective agonist ONO-AE1-329, mimicked the effects of PGE(2) on phagocytosis and cAMP stimulation. Additionally, the EP2 antagonist AH-6809 abrogated the inhibitory effects of both PGE(2) and butaprost. We confirmed the specificity of our results by showing that AMs from EP2-deficient mice were resistant to the inhibitory effects of PGE(2). Our data support a negative regulatory role for PGE(2) on the antimicrobial activity of AMs, which has important implications for future efforts to prevent and treat bacterial pneumonia.     

Prostaglandin E(2) and stem cell factor can deliver opposing signals to B lymphocyte precursors

The synthetic prostanoid, 16,16-dimethyl PGE(2), suppressed B lymphopoiesis in mice and proliferation of normal B cell precursors or the F10 pro-B cell line to interleukin 7 in culture. This was not the case with two other prostanoids, PGD(2) and PGF(2alpha), or agonists for PGI(2) agonist and thromboxane A(2) agonist receptors. PGE(2), but not the related prostanoids or agonists, induced apoptosis in F10 cells. The apoptotic response was mediated by the EP2 class of PGE(2) receptors and required an increase in intracellular cyclic adenosine 3',5'-monophosphate, activation of protein kinase A, and protein synthesis. The influence of PGE(2) on F10 cells was diminished in the presence of a cloned stromal cell line or stem cell factor. These findings describe another potential regulatory circuit in bone marrow which might influence B lymphopoiesis under disease or steady-state conditions.     

Prostaglandin E2 enhances interleukin-8 production via EP4 receptor in human pulmonary microvascular endothelial cells

Prostaglandin E(2) (PGE(2)) is a bioactive prostanoid implicated in the inflammatory processes of acute lung injury/acute respiratory distress syndrome. This study investigated whether PGE(2) can induce production of interleukin (IL)-8, the major chemokine for neutrophil activation, from human pulmonary microvascular endothelial cells (HPMVECs). PGE(2) significantly enhanced IL-8 protein production with increases in IL-8 mRNA expression and intracellular cAMP levels. HPMVECs expressed only EP4 receptor mRNA. The PGE(2) effects were mimicked by a selective EP4 receptor agonist, ONO-AE1-329, and inhibited by a selective EP4 receptor antagonist, ONO-AE3-208, or a protein kinase A inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt. The specific agonist for EP1, EP2, or EP3 receptor did not induce IL-8 production. PGE(2)-induced IL-8 production was accompanied by p38 phosphorylation and was significantly inhibited by a p38 inhibitor, SB-203580, but not by an ERK1/2 inhibitor, U-0126, or a JNK inhibitor, SP-600125. Additionally, PGE(2) increased cyclooxygenase-2 expression with no change in constitutive cyclooxygenase-1 expression, suggesting possible involvement of an autocrine or paracrine manner. In conclusion, PGE(2) enhances IL-8 production via EP4 receptor coupled to G(s) protein in HPMVECs. Activation of the cAMP/protein kinase A pathway, followed by p38 activation, is essential for these mechanisms. Because neutrophils play a critical role in the inflammation of acute lung injury/acute respiratory distress syndrome, IL-8 released from the pulmonary microvasculature in response to PGE(2) may contribute to pathophysiology of this disease.     

Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes,EP3 and EP4

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.     

The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages

The expression and regulation of the PGE receptors, EP(2) and EP(4), both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP(4) but not EP(2) was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP(4) was down-regulated by LPS but not by medium change. PGE(2) increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP(4) agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP(2) agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-alpha and IL-12 production were equipotent with PGE(2) at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE(2) or dibutyryl cAMP alone, but not butaprost, reduced EP(4) expression, and indomethacin reversed the LPS-induced down-regulation of EP(4), indicating that the down-regulation of EP(4) is mediated by LPS-induced PG synthesis and EP(4) activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP(4) expression was found in the cells treated with PGE(2) alone, but not in LPS-treated cells. In contrast, up-regulation of EP(2) expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP(4) is involved mainly in the inhibition of cytokine release, and that the gene expression of EP(2) and EP(4) is differentially regulated during macrophage activation.     

Cyclooxygenase-2 expression in lipopolysaccharide-stimulated human monocytes is modulated by cyclic AMP, prostaglandin E(2), and nonsteroidal anti-inflammatory drugs

Using human blood monocytes (for determination of cyclooxygenase-2 (COX-2) mRNA by RT-PCR) and human whole blood (for prostanoid determination), the present study investigates the influence of the second messenger cAMP on lipopolysaccharide (LPS)-induced COX-2 expression with particular emphasis on the role of prostaglandin E(2) (PGE(2)) in this process. Elevation of intracellular cAMP with a cell-permeable cAMP analogue (dibutyryl cAMP), an adenylyl cyclase activator (cholera toxin), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) substantially enhanced LPS-induced PGE(2) formation and COX-2 mRNA expression, but did not modify COX-2 enzyme activity. Moreover, up-regulation of LPS-induced COX-2 expression was caused by PGE(2), butaprost (selective agonist of the adenylyl cyclase-coupled EP(2) receptor) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist), whereas sulprostone (EP(3)/EP(1) agonist) left COX-2 expression unaltered. Abrogation of LPS-induced PGE(2) synthesis with the selective COX-2 inhibitor NS-398 caused a decrease in COX-2 mRNA levels that was restored by exogenous PGE(2) and mimicked by S(+)-flurbiprofen and ketoprofen. Overall, these results indicate a modulatory role of cAMP in the regulation of COX-2 expression. PGE(2), a cAMP-elevating final product of the COX-2 pathway, may autoregulate COX-2 expression in human monocytes via a positive feedback mechanism.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.     

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.