Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.     

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.     

Role of prostaglandin E(2) EP receptors and cAMP in the expression of connective tissue growth factor

Wild-type (WT) Rat-1 fibroblasts express undetectable quantities of the prostaglandin E(2) (PGE(2)) EP1, EP2, and EP3 receptor types and detectable amounts of the EP4 receptor. In the WT Rat-1, PGE(2) enhances connective tissue growth factor (CTGF) mRNA. PGE(2) does not stimulate cAMP production in these cells. However, forskolin induces cAMP production and ablates TGF beta-stimulated increases in CTGF mRNA. A similar pattern of CTGF expression in response to PGE(2) and forskolin is observed in neonatal rat primary smooth muscle cell cultures. When WT Rat-1 cells are stably transfected with the EP2 receptor, PGE(2) causes a sizable elevation in intracellular cAMP and ablates the TGF beta-stimulated increase in CTGF mRNA. PGE(2) does not have this effect on cells expressing the EP1, EP3, or EP4 receptor subtypes. These results demonstrate the importance of the EP2 receptor and cAMP in the inhibition of TGF beta-stimulated CTGF production and suggest a role for PGE(2) in increasing CTGF mRNA levels in the absence of the EP2 receptor. Involvement of inositol phosphate in this upregulation of CTGF expression by PGE(2) is doubtful. None of the cell lines containing the four EP transfectants nor the WT Rat-1 cells responded to PGE(2) with inositol phosphate production.     

Prostaglandin E(2) inhibits IL-18-induced ICAM-1 and B7.2 expression through EP2/EP4 receptors in human peripheral blood mononuclear cells

Costimulatory molecules play important roles in immune responses. In the present study we investigated the effects of PGE(2) on the expression of ICAM-1, B7.1, and B7.2 on monocytes in IL-18-stimulated PBMC using FACS analysis. Addition of PGE(2) to PBMC inhibited ICAM-1 and B7.2 expression elicited by IL-18 in a concentration-dependent manner. We examined the involvement of four subtypes of PGE(2) receptors, EP1, EP2, EP3, and EP4, in the modulatory effect of PGE(2) on ICAM-1 and B7.2 expression elicited by IL-18, using subtype-specific agonists. ONO-AE1-259-01 (EP2R agonist) inhibited IL-18-elicited ICAM-1 and B7.2 expression in a concentration-dependent manner with a potency slightly less than that of PGE(2), while ONO-AE1-329 (EP4R agonist) was much less potent than PGE(2). The EP2/EP4R agonist 11-deoxy-PGE(1) mimicked the effect of PGE(2) with the same potency. ONO-D1-004 (EP1R agonist) and ONO-AE-248 (EP3R agonist) showed no effect on IL-18-elicited ICAM-1 or B7.2 expression. These results indicated that EP2 and EP4Rs were involved in the action of PGE(2). Dibutyryl cAMP and forskolin down-regulated ICAM-1 and B7.2 expression in IL-18-stimulated monocytes. As EP2 and EP4Rs are coupled to adenylate cyclase, we suggest that PGE(2) down-regulates IL-18-induced ICAM-1 and B7.2 expression in monocytes via EP2 and EP4Rs by cAMP-dependent signaling pathways. The fact that anti-B7.2 as well as anti-ICAM-1 Ab inhibited IL-18-induced cytokine production implies that PGE(2) may modulate the immune response through regulation of the expression of particular adhesion molecules on monocytes via EP2 and EP4Rs.     

Role of cyclic adenosine 3',5'-monophosphate in mediating the effect of prostaglandin E2 on decidualization in vitro.

When rat endometrial stromal cells from uteri sensitized for decidualization are cultured in vitro, there is an increase in alkaline phosphatase (ALP) activity paralleling that seen in vivo during decidualization. The addition of indomethacin to the culture medium decreases the endogenous production of prostaglandin E2 (PGE2) to below detectable levels and substantially reduces the increase in ALP activity. The addition of either PGE2 or its analog 16,16-dimethyl-PGE2, but not PGF2 alpha or its analog 15(S),15-methyl-PGF2 alpha, overrides this inhibitory effect, suggesting that PGE2 has a specific stimulatory effect upon ALP activity. This in vitro system was used to investigate the role of the cAMP pathway in mediating the stimulatory effect of PGE2 on ALP activity. The data indicate that PGE2 causes an increase in cAMP accumulation by the cells and that the addition of an analog of cAMP or substances which increase the level of cAMP in the cells (1-methyl-3-isobutyl xanthine, cholera toxin, forskolin) causes an increase in ALP activity. Collectively, the results suggest that the stimulatory effect of PGE2 is at least partially mediated by the cAMP pathway.     

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.     

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.     

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E(1) (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 microM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.     

Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.     

Administration of parathyroid hormone, prostaglandin E2, or 1-alpha,25-dihydroxyvitamin D3 restores the bone inductive activity of rhBMP-2 in aged rats

Bone morphogenetic protein (BMP) induces bone formation in young rodents, but aging causes a reduction in the bone-forming ability of BMP. Most patients who require bone reconstruction are relatively old. Accordingly, we examined whether anabolic hormones could restore the bone inductive activity of rhBMP-2 in aged rats. rhBMP-2 in a carrier pellet was implanted subcutaneously in both 4- and 50-week-old female Wistar rats. PTH, PGE2, or 1,25(OH)2D3 was injected every day during the period of BMP implantation. The pellets were harvested, and were examined both histologically and biochemically 2 weeks after implantation. Bone-forming ability was measured by alkaline phosphatase (ALP) activity and calcium (Ca) content. Pellets in 50-week-old rats showed a significant reduction in bone formation compared to pellets in 4-week-old rats. However, daily injections of PTH into 50-week-old rats restored both ALP activity (103 +/- 4.6%) and Ca content (105 +/- 2.6%). 1,25(OH)2D3 and PGE2 also restored Ca content (103 +/- 4.5% and 98 +/- 3.8%, respectively) and stimulated ALP activity (142 +/- 2.3% and 133 +/- 3.6%). These results show that the administration of these hormones restores bone-forming ability in aged rats. A combination treatment of these hormones with rhBMP-2 might be applicable to the reconstruction of bone defects in elderly patients.     

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.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.     

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.     

PGE(2) receptors and their intracellular mechanisms in rabbit small intestine

The effects of PGE(2) on longitudinal smooth muscle, the intracellular mechanisms involved, and the localization of EP receptors were investigated in rabbit small intestine. PGE(2) evoked contractions in small intestine that were reduced by tetrodotoxin and hexamethonium. 17-Phenyl trinor PGE(2), sulprostone, misoprostol and 16,16-dimethyl PGE(2) evoked contractions. Butaprost did not modify spontaneous motility. AH 6809 reduced PGE(2) and 17-phenyl trinor PGE(2)-induced contractions. Verapamil, Ca(2+) free medium, staurosporine, forskolin, theophylline, and rolipram diminished, while IP-20 and H-89 increased PGE(2)-induced contractions. Western blot analysis showed protein bands of 41kDa for EP(1), 71kDa for EP(2) and 62kDa for EP(3) receptors. EP(1), EP(2) and EP(3) receptors were detected in neurons of the myenteric and submucosal ganglia, but only EP(3) receptors were found in smooth muscle layers. This study did not detect EP(4) receptor. PGE(2)-induced contractions would be mediated through EP(1) and EP(3) receptors, and voltage-dependent Ca(2+) channels, protein kinase C, and cAMP would be implicated in these responses.     

Potentiation of PGE2-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.     

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs

Prostaglandin E2 (PGE2)-dependent effects on various cell responses are regulated by respective PGE2 receptors (EP1, EP2, EP3, EP4) expressing in target cells. Alveolar type II cell (a main progenitor cell of lung adenocarcinoma) expressed only EP4, while human lung adenocarcinoma cells (A549) expressed EP3 as well as EP4. An antagonistic effect of EP3 against EP4 through the modulation of cyclic AMP level is required for PGE2-mediated activation of Ras signal pathway in A549 cells. These results suggest that the expression of EP3 may be a critical factor for the PGE2-mediated activation of Ras signal pathway in A549 cells.     

Effect of prostaglandin E2 on PMA-induced macrophage differentiation

Major trauma such as severe bums and extensive surgery could result in accelerated macrophage differentiation and hyperactivation causing an excessive release of proinflammatory cytokines and prostaglandin E2 (PGE2) with consequent severe impairment of immunologic reactivity. HL-60 cells stimulated with phorbol 12-myristate 13-acetate (PMA) have been used as a model to asses the PGE2 role in the macrophage differentiation observed after major trauma. Cell adhesion, matrix metalloproteinase-9 (MMP-9) and tumor necrosis factor-alpha (TNF-alpha) production were measured after 24 h of PMA treatment in the presence of PGE2 (1 nM - 1 microM). PGE2 increased both the PMA-induced cell adhesion and MMP-9 production via EP2/EP4 receptors while it had no effect on the induced TNF-alpha release. The cAMP/PKA pathway, usually linked to EP2/EP4 activation, was not involved in the phenomenon, suggesting that an alternative signalling pathway could be linked to a PKC-activated enzyme. In fact PGE2 activity was partially inhibited by Wortmannin, a phosphoinositide-3 kinase (PI-3K) inhibitor indicating that PGE2 act as a co-factor able to increase macrophage differentiation in vitro via a PI-3K dependent pathway that could be also involved in the immunosuppression observed in the aftermath of trauma.