Unique suppression of prostaglandin H synthase-2 expression by inhibition of histone deacetylation, specifically in human amnion but not adjacent choriodecidua

The key molecular regulatory mechanisms that govern and coordinate the molecular alterations that underpin the process of human labor remain incompletely understood although enhanced intrauterine prostaglandin production is known to be requisite. Studies from cancer tissues have indicated that at least one key enzyme of prostaglandin biosynthesis can have its activity severely reduced by increased histone deacetylation and enhanced DNA methylation status. We have advanced the hypothesis that similar regulation may occur in intrauterine tissues during pregnancy to prevent inadvertent activation of this powerful initiating signal by dampening responses to premature activation by agents such as cytokines. Our studies have shown that responsiveness of amnion, a key intrauterine tissue, to interleukin-1beta is abrogated by inhibition of histone deacetylation, whereas PGDH amounts were increased basally. The findings do integrate well with others concerning progesterone (inhibitory) actions such that a decrease in the level of histone acetylation in human gestational tissues near term might herald a coordinated series of events that all result in a positive drive for parturition. Hence, a new level of regulatory action and potential therapeutic targets for pathologies such as preterm labor can flow from these findings.     

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.     

Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages

Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E₂ (PGE₂). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE₂ and prostaglandin H synthase-2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL-4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS-binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE₂, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. © 1995 Wiley-Liss Inc.     

Corticotropin-releasing hormone increases the expression of the prostaglandin E(2) receptor subtype EP1 in amnion WISH cells

The purpose of this study was to investigate the effect of corticotropin-releasing hormone (CRH) on the expression of the prostaglandin (PG) E(2) EP1 receptor subtype and PGE(2) production in amnion WISH cells (AWC). AWC cultures were incubated with CRH. Culture fluid was collected for PGE(2) measurement, and the cells were collected and analyzed for EP1 protein and mRNA. Immunohistochemical localization of the EP1 receptor was also performed. Incubation of AWC with CRH resulted in a dose-dependent increase (r = 0.97) in the level of EP1 receptor protein (P < 0.001). Coincubation of AWC with CRH and indomethacin resulted in the decreased production of PGE(2) while having no effect on EP1 receptor expression. A significant but not dose-dependent increase in EP1 mRNA expression was also observed (P < 0.01). Immunohistochemical evaluation verified cell membrane localization of the receptor in both stimulated and unstimulated cells and confirmed the increased expression of EP1 receptor in response to CRH. Incubation of AWC with CRH also resulted in increased culture fluid PGE(2) levels (P < 0.01). These results suggest that the role CRH plays in the initiation of labor may also involve the promotion of elevated PGE(2) levels and increased expression of the EP1 receptor in amnion.     

Paradoxical stimulation of both lipocortin and prostaglandin production in human amnion cells by dexamethasone

Glucocorticoids inhibit prostaglandin biosynthesis by inducing the formation of lipocortins. In human amnion cells dexamethasone elicited a concentration-dependent increase in prostaglandin production and raised intracellular lipocortin 1 concentrations. Dexamethasone could also potentiate the epidermal growth factor (EGF)-induced stimulation of prostaglandin production. EGF alone or in combination with dexamethasone increased lipocortin 1 formation in amnion cells. Human amnion cells may provide a unique insight into interactions between glucocorticoids, lipocortin and eicosanoid biosynthesis.     

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.     

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI(2) , acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE(2) and PGI(2) in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF(1-2) , without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE(2) acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.     

Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Tyrosine kinase inhibitors block the glucocorticoid stimulation of prostaglandin endoperoxide H synthase expression in amnion cells.

Human amnion cells in primary culture respond to glucocorticoids in a characteristic fashion by the increased expression of the inducible prostaglandin endoperoxide H synthase isoenzyme, PGHS-2. Since PGHS-2 induction by agonists generally involves tyrosine kinases, we examined the possibility that the glucocorticoid stimulation of PGHS-2 in the amnion cells is tyrosine kinase dependent. PGHS-2 expression was stimulated in confluent, serum-starved amnion cells with dexamethasone, and the effect of the tyrosine kinase inhibitors herbimycin A and tyrphostins AG126, AG1288, and A1 on enzyme activity induction was determined. All four inhibitors blocked the increase of PGHS activity in a concentration-dependent manner with IC50 values of 0.077 +/- 0.05, 15.38 +/- 5.14, 20.91 +/- 3.1, and 29.77 +/- 8.21 microM, respectively (mean +/- SE, n = 4). Dexamethasone increased (approximately twofold) the tyrosine phosphorylation of 120-, 110-, and 77-kDa proteins in cell extracts, and herbimycin A selectively blocked the phosphorylation of the 110-kDa phosphoprotein. The stimulation of the steady-state level of PGHS-2 mRNA by dexamethasone was also inhibited by herbimycin A. These results suggest that glucocorticoids induce PGHS-2 expression in amnion cells with the involvement of tyrosine kinase(s). The role of tyrosine kinase dependent mechanisms in the control of amnion cell responsiveness to corticosteroids remains to be established.     

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.