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 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2). PGE(2) led to an increase of COX-2 mRNA and protein expression, whereas the expression of COX-1 remained unchanged. Upregulation of COX-2 expression by PGE(2) was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, and was abrogated by inhibitors of both pathways. Moreover, PGE(2)-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II, whereas the protein kinase A inhibitor H-89 was inactive in this respect. Induction of COX-2 expression was also elicited by butaprost (EP(2) receptor agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) receptor agonist), but not by EP(1)/EP(3) receptor agonists (17-phenyl-omega-trinor PGE(2), sulprostone). Consistent with these findings, the EP(1)/EP(2) receptor antagonist, AH-6809, and the selective EP(4) receptor antagonist, ONO-AE3-208, significantly reduced PGE(2)-induced COX-2 expression. Collectively, our results demonstrate that PGE(2) at physiologically relevant concentrations induces COX-2 expression in human NPE cells via activation of EP(2)- and EP(4) receptors and phosphorylation of p38 and p42/44 MAPKs. Positive feedback regulation of COX-2 may contribute to the production of outflow-facilitating PGs and consequently to regulation of IOP.     

Prostaglandin E2 suppresses lipopolysaccharide-stimulated IFN-beta production

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.     

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.     

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.     

Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages

COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1-4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors.     

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.     

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.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Macrolide antibiotics promote the LPS-induced upregulation of prostaglandin E receptor EP2 and thus attenuate macrolide suppression of IL-6 production

We studied the influence of the inhibitory effect of clarithromycin (CAM) and erythromycin (EM) on the production of macrophage inflammatory protein (MIP)-2, interleukin-6 (IL-6), and prostaglandin E(2) (PGE(2)), as well as PGE(2) receptor (EP(2)) expression, by LPS-stimulated RAW264.7 cells. Production of IL-6 was significantly decreased by treatment with CAM or EM in a dose-dependent manner, but the inhibitory effect of CAM was significantly weaker than that of EM. In contrast, the production of MIP-2 and PGE(2) was inhibited to the same extent by CAM and EM. LPS induced the expression of EP(2) mRNA and its expression was promoted further by treatment with CAM or EM. In particular, CAM significantly upregulated EP(2) mRNA expression compared with that after stimulation by LPS alone. After treatment with a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), or an EP(2)/EP(4) receptor antagonist (AH6809), the inhibitory effect of CAM and EM on LPS-induced IL-6 production was equalized. These results indicate that macrolide antibiotics upregulate the expression of EP(2), which then attenuates the suppressive effect on IL-6 production of these antibiotics, suggesting that these drugs have a variable anti-inflammatory effect that could influence host defenses.     

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.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

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.     

Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44- and MMP-2-mediated invasion in NSCLC in an autocrine/paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.     

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.