Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E2 is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E2 by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E2 than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E2; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E2 biosynthesis in this tissue.     

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.     

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.     

Effects of glucocorticoids on prostaglandin formation by human amnion

The human amnion may be an important source of prostaglandins involved in the onset of human labor and therefore it is important to define the factors that regulate their formation in this tissue. In the present study we demonstrate that glucocorticoids inhibit prostaglandin production by freshly isolated amnion cells. The inhibitory action of the glucocorticoids, however, changes to a stimulatory action when the cells are maintained in primary culture for a few days. For both inhibition and stimulation, concentrations of 10(-8) M dexamethasone or greater were required to give significant effects, and estradiol and progesterone had no effect on the prostaglandin output of the cells. Epidermal growth factor (EGF), which has previously been found to stimulate prostaglandin output by confluent amnion cells, did not alter prostaglandin output of cells initially placed in culture. Furthermore, the stimulatory action of EGF and dexamethasone appeared additive. The calcium ionophore A23187 stimulated prostaglandin output in freshly isolated cells and accentuated the inhibitory effect of dexamethasone. These studies indicate that prostaglandin formation by human amnion during pregnancy could be regulated by glucocorticoids. These steroids are easily available to the amnion by way of cortisone conversion to cortisol by the maternal decidua. The results also indicate that amnion is capable of responding to glucocorticoids in both a stimulatory and inhibitory fashion and whether one or both actions are of importance in vivo is a question that is as yet unresolved.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained in vitro were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E2. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E2 release on the concentration of isoproterenol were similar, each response was maximal at 10(-6) M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E2 from amnion discs. Although prostaglandin E2, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, may be regulators of prostaglandin production by the amnion.     

Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid.

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E2 production as well as a smaller increase in non-cyclo-oxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E2 production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F2 alpha, prostacyclin and thromboxane A2. Prostaglandins E2 and F2 alpha are the principal cyclo-oxygenase products of this interaction. We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F2 alpha by leukocytes.     

A monokine stimulates prostaglandin-E2 production by human amnion

The studies reported in this communication were designed to test the hypothesis that products of mononuclear cells are capable of stimulating prostaglandin E2 production by human amnion. Conditioned media obtained from peripheral blood mononuclear cells were incubated with amnion cells in primary culture. A dose dependent increase in PGE2 biosynthesis was observed in response to increasing amounts of the conditioned media. These observations suggest that mononuclear cells produce a factor(s) capable of stimulating prostaglandin production by amnion cells. The signal responsible for the increased biosynthesis of prostaglandins by human amnion associated with parturition in the setting of intraamniotic infection may be of host origin.     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.     

Platelet-activating factor metabolism in human amnion and the responses of this tissue to extracellular platelet-activating factor

It was found previously that platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is undetectable in human amniotic fluid obtained before labor but is present in the majority of samples of amniotic fluid obtained after labor. In the present investigation, the amount of PAF in amnion tissue and the ability of this tissue to produce PAF and respond to PAF were investigated. Amounts of PAF in amnion obtained either during the second trimester of gestation or at term (before labor) were similar. After labor, however, the amount of PAF in amnion increased to 2.5-times that in amnion before labor without any discernible changes in the amounts of two related glycerophospholipids viz., 1-0-alkyl-sn-glycero-3-phosphocholine and 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine. The Ca2+-ionophore A23187, in the presence of Ca2+, caused an increase in the amount of PAF in amnion tissue disks but PAF did not appear to be released into the incubation medium. The stimulation of PAF formation by A23187 and Ca2+ was not affected by the addition of indomethacin. Addition of PAF to disks of amnion tissue resulted in an increase in the concentration of prostaglandin E2 in the incubation medium. An increase in prostaglandin E2 formation of similar magnitude was induced by A23187. Based on these results it is concluded that PAF can be synthesized in amnion tissue and net production is stimulated by Ca2+. In addition, amnion is receptive to extracellular PAF and exhibits, as one response, an increased production of prostaglandin E2.     

Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E₂ is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E₂ by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E₂ than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E₂; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E₂ biosynthesis in this tissue.     

Interleukin-4 differentially regulates prostaglandin production in amnion-derived WISH cells stimulated with pro-inflammatory cytokines and epidermal growth factor.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.     

Amnion cell prostaglandin E2 stimulatory activity in chorion laeve-conditioned medium

In this study we demonstrate the presence of a stimulant(s) to amnion cell prostaglandin (PG) E2 production in chorion-conditioned medium (CCM). The CCM induced a dose-dependent increase in amnion cell PGE2 production. This stimulatory activity was eliminated by heat and protease treatment. Maximal stimulation of amnion PGE2 by CCM did not occur until after 2 h of incubation, and treatment with cycloheximide (1 microgram/ml) effectively eliminated the ability of the amnion cells to respond to CCM. Additionally, CCM and arachidonic acid (2-40 microM) were synergistic in their stimulatory actions on amnion PGE2 production. CCM-treated amnion cells recover more quickly from acetylsalicylic acid pretreatment as compared to control. It is concluded that CCM contains a heat-labile protein which stimulates amnion cell PGE2 production by induction of prostaglandin endoperoxide synthase activity.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Labor and inflammation increase the expression of oxytocin receptor in human amnion

The oxytocin/oxytocin receptor (OXT/OXTR) system plays an important role in the regulation of parturition. The amnion is a major source of prostaglandins and inflammatory cytokine synthesis, which increase both before and during labor. Amnion is a noncontractile tissue; therefore, the role played by OXT/OXTR in this tissue will be fundamentally different from the role played in myometrial contractions. In the present study, we demonstrate increased OXTR mRNA and protein concentrations in human amnion epithelial cells associated with the onset of labor. We show that incubation of primary human amnion epithelial cells with IL1B results in a rapid, transient up-regulation of OXTR mRNA expression, which peaks in prelabor samples after 6 h. Incubation of prelabor amnion epithelial cells with OXT results in a marked increase of prostaglandin E(2) synthesis, and we demonstrate that OXT activates the extracellular signal-regulated protein kinase signal transduction pathway to stimulate up-regulation of cyclo-oxygenase 2 in human amnion epithelial cells. The increased ability of human amnion to produce prostaglandins in response to OXT treatment suggests a complementary role for the OXT/OXTR system in the activation of human amnion and the onset of labor.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E₂ production as well as a smaller increase in non-cyclooxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E₂ production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F_2α, prostacyclin and thromboxane A₂. Prostaglandins E₂ and F_2α are the principal cyclo-oxygenase products of this interaction.We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F_2α by leukocytes.