Increased progesterone receptor A expression in labouring human myometrium is associated with decreased promoter occupancy by the histone demethylase JARID1A.

Progesterone regulates female reproductive function predominantly through two nuclear progesterone receptors (PRs), PR-A and PR-B. During human parturition myometrial PR expression is altered to favour PR-A, which activates pro-labour genes. We have previously identified histone H3 lysine 4 trimethylation (H3K4me3) as an activator of myometrial PR-A expression at labour. To further elucidate the mechanisms regulating PR isoform expression in the human uterus at labour, we have (i) determined the methylation profile of the cytosine-guanine dinucleotides (CpG) island in the promoter region of the PR gene and (ii) identified the histone-modifying enzymes that target the H3K4me3 mark at the PR promoters in term and preterm human myometrial tissues obtained before and after labour onset. Bisulphite sequencing showed that despite overall low levels of PR CpG island methylation, there was a significant decrease in methylated CpGs with labour in both preterm (P < 0.05) and term (P < 0.01) groups downstream of the PR-B transcription start site. This methylation change was not associated with altered PR-B expression, but may contribute to the increase in PR-A expression with labour. Chromatin immunoprecipitation revealed that the histone methyltransferase, SET and MYND domain-containing protein 3 (SMYD3), bound to the PR gene at significantly higher levels at the PR-A promoter compared with the PR-B promoter (P < 0.010), with no labour-associated changes observed. The H3K4 demethylase, Jumonji AT-rich interactive domain 1A (JARID1A), also bound to the PR-A, but not to the PR-B promoter prior to term labour, and decreased significantly at the onset of labour (P = 0.014), providing a mechanism for the previously reported increase in H3K4me3 level and PR-A expression with labour. Our studies suggest that epigenetic changes mediated by JARID1A, SMYD3 and DNA methylation may be responsible, at least in part, for the functional progesterone withdrawal that precipitates human labour.

Regulation of the renin-angiotensin system (RAS) in BeWo and HTR-8/SVneo trophoblast cell lines.

OBJECTIVES: The renin-angiotensin system (RAS) is implicated in placentation. We determined which RAS pathways are present in two trophoblast cell lines (HTR-8/SVneo and BeWo cells) and the effects of cAMP, which stimulates renal renin. STUDY DESIGN: The effect of cAMP on RAS gene expression and on prorenin and angiotensin peptides in HTR-8/SVneo and BeWo cells were investigated. RESULTS: In HTR-8/SVneo cells, prorenin mRNA (REN) and protein, (pro)renin receptor (ATP6AP2) and angiotensin II type 1 receptor (AGTR1) were stimulated by cAMP (P < 0.05, P < 0.05, P < 0.001 and P < 0.05, respectively). HTR-8/SVneo cells also expressed angiotensinogen (AGT) and angiotensin converting enzyme 1 (ACE1), but did not express AGTR2 or ACE2 nor the Ang 1-7 receptor (MAS1). BeWo cells did not express REN, and REN was not inducible by cAMP, but cAMP increased ACE2 and MAS1 (both P < 0.05) and decreased AGT (P < 0.05). BeWo cells expressed AGT, ACE1, ACE2 and MAS1 but not ATP6AP2, AGTR1 nor AGTR2. There was net destruction of Ang II in media from HTR-8/SVneo and BeWo incubations and net production of Ang 1-7 by BeWo and untreated HTR-8/SVneo cells. CONCLUSION: HTR-8/SVneo cells express REN and produce prorenin as well as expressing other RAS genes likely to regulate Ang II/AT(1)R interactions and respond to cAMP, like renal renin-secreting cells. They are more similar to early gestation placentae and are therefore useful for studying effects of renin/ACE/Ang II/AT1R on cell function. BeWo cells express the ACE2/Ang 1-7/Mas pathway, which is sensitive to cAMP and therefore are useful for studying the effects of ACE2/Ang 1-7/Mas on trophoblast function.

Term myometrium is characterized by increased activating epigenetic modifications at the progesterone receptor-A promoter.

Term human myometrial expression of progesterone receptor (PR)-A is increased relative to PR-B, and as PR-A is a repressor of progesterone action mediated through PR-B, this increase may mediate the withdrawal of progesterone action and precipitate the onset of labour. PR-A and PR-B expression is regulated by two separate promoters of the PR gene. We hypothesized that epigenetic histone modifications at the two promoters contribute to the labour-associated regulation of PR-A and PR-B expression in term myometrium. PR total, PR-B and PR-A mRNA levels were determined using quantitative real-time PCR, and chromatin immunoprecipitation was used to determine the levels of activating and repressive histone modifications at the PR-A and PR-B promoters in human myometrial samples not in labour (n = 4) and in labour (n = 4). Chromatin extracts were immunoprecipitated with antibodies against activating (histone H3 and H4 acetylation and histone H3 lysine 4 trimethylation), and repressive (histone H3 lysine 9 trimethylation, histone H3 lysine 27 trimethylation and asymmetrical histone H3 arginine 2 dimethylation) histone modifications. PR-A mRNA levels increased during labour, while PR-B mRNA levels remained constant resulting in an increase of PR-A/PR-B mRNA ratio, as expected. Regardless of labour status, significantly higher levels of the activating histone modifications were found at the PR-A promoter compared with the PR-B promoter (P <0.001). H3K4me3 increased significantly at both promoters with labour onset (P =0.001). Low levels of the repressive histone modifications were also present at both promoters, with no labour-associated changes observed. Our data indicate that the PR-A promoter is epigenetically marked for activation in term myometrium more extensively than the PR-B promoter, and that labour is associated with an increase in H3K4me3 activating modification, consistent with the previously described increase in PR protein at this time.

Molecular characterization of renin-angiotensin system components in human intrauterine tissues and fetal membranes from vaginal delivery and cesarean section.

A prorenin-angiotensin system (RAS) could, via the (pro)renin receptor (ATP6AP2), have various effects in human intrauterine tissues, either directly by prorenin/ATP6AP2 cell signaling, or indirectly via angiotensin II and/or angiotensin 1-7. Here we describe RAS components in fetal membranes, decidua and placenta collected at elective cesarean section (non-laboring), after spontaneous delivery (after labor, n = 38), and in myometria (n = 16) from elective (non-laboring) or emergency cesarean (laboring) deliveries. Angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin receptor 1 and 2 (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1) mRNAs were measured by qRT-PCR and proteins were localized by immunohistochemistry. In myometrium, prorenin (REN), ATP6AP2, and downstream signaling proteins zinc finger and BTB domain-containing protein 16 (ZBTB16), transforming growth factor-ß1 (TGFß1) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs were also measured. RAS mRNAs, except AGTR1 and AGTR2, were abundant in decidua and lowest in amnion compared to the other tissues. ACE, AGT and PTGS2 mRNAs were higher in laboring than non-laboring myometrium, suggesting that the myometrial RAS is involved in labor. Angiotensinogen and prorenin staining in amnion, chorion and decidua was pervasive despite their mRNAs being low in amnion and chorion. In placenta, prorenin, angiotensinogen and AGTR2 were present in syncytiotrophoblasts, ACE was in fetal endothelium, while ACE2 distribution was diffuse. AGTR1 and AGTR2 mRNAs and proteins were abundant. No differences were evident in the staining patterns with labor. These results are consistent with the hypothesis that fetal vascular ACE might contribute angiotensin II to the fetus, whilst syncytial ACE2 might hypothetically have a role in converting angiotensin II to angiotensin 1-7 in maternal blood.

Prostaglandin H synthase-2 gene regulation in the amnion at labour: histone acetylation and nuclear factor kappa B binding to the promoter in vivo.

Increased prostaglandin H synthase-2 (PGHS-2) expression in the amnion is critical for the production of prostaglandins that induce labour. The aim of the present investigation was to determine whether PGHS-2 gene activity is controlled by NFkappaB transcription factors in term amnion in vivo as suggested by in vitro findings. Amnion membranes were collected after elective Caesarean section (n = 14) or spontaneous labour (n = 12) at term, and histone acetylation and transcription factor binding to the PGHS-2 and IkappaBalpha promoters were determined in fresh tissues by chromatin immunoprecipitation. High level of histone-3 and -4 acetylation was detected in the proximal 1000 bp region of the PGHS-2 promoter indicating permissive chromatin structure in an area that contains two consensus NFkappaB binding sites and other transcription factor binding motifs. The TATA-box was occupied by TATA-binding protein (TBP) demonstrating that the PGHS-2 gene was transcriptionally active before and after labour. NFkappaB (p65 and p50) binding to the consensus sites, however, was detected only before, but not after, labour. Moreover, NFkappaB factor binding before labour was unrelated to TBP binding to the PGHS-2 TATA-box in the same tissues. Further, p65 binding to the NFkappaB-responsive IkappaBalpha promoter increased at labour and correlated strongly with TBP binding to the TATA-box of this gene. We conclude that the proximal 1000 bp region is involved in PGHS-2 promoter regulation in term amnion. The NFkappaB system is activated at labour and stimulates the IkappaBalpha gene, but the NFkappaB factors do not drive PGHS-2 transcription using consensus promoter sites in normal term amnion in vivo.

Regulation of myometrial smooth muscle functions.

Regulation of myometrial functions during gestation, labor and birth are in the forefront of research in reproductive sciences. The complexity of the problem is reflected by our scant understanding of the intimate cellular and molecular events underlying these phenomena, despite extensive efforts spanning several decades. Unlike other smooth muscles, the myometrium is, to a large extent, under hormonal control. Of these, the steroid hormones, progesterone and estrogen, play dominant roles in terms of uterine growth, the maintenance of quiescence during gestation and the preparation of the uterus for labor and delivery. In addition to steroid hormones, there are a number of factors that modulate myometrial contractility (oxytocin, prostaglandins, endothelin, platelet activating factor) and relaxation (corticotropin releasing hormone, prostacyclin, nitric oxide). Although notable advances have been made towards understanding some of the key steps in receptor signaling that define the actions of these factors, a good deal of new information is needed to fully understand this fundamental life process. Pharmaceutical agents have been used extensively to induce labor or to prolong pregnancy in the case of preterm labor that represents the major cause of perinatal morbidity and mortality. Because preterm labor is a syndrome of multiple etiologies, pharmacologic agents will have to be targeted accordingly. This review attempts to present a critical overview of these topics.

Regulation of prostaglandin synthesis in the human uterus.

Prostaglandins are important regulators of many aspects of reproductive processes from ovulation, fertilization and pregnancy recognition to labor and parturition. These biologically potent compounds are members of the large family of eicosanoids, derived from polyunsaturated fatty acids, principally arachidonic acid, found in the membrane phospholipids of virtually every cell of the human body, accounting for the ubiquity of prostaglandins, which act in a paracrine or autocrine fashion via discrete receptors. The availability of specific prostaglandins in various cells and tissues depends on the presence and activity of specific enzymes that convert a common precursor to the end product, as well as on the rate of enzymatic or spontaneous inactivation of the bioactive compounds. Here we offer a brief review of the regulation of prostaglandin generation in human uterine tissues, focusing on their role in labor and parturition at term and preterm.

Prostaglandin endoperoxide H synthase mRNA expression in the fetal membranes correlates with fetal fibronectin concentration in the cervico-vaginal fluids at term: evidence of enzyme induction before the onset of labour.

OBJECTIVE: To determine the relationship of prostaglandin endoperoxide H synthase (PGHS) expression in the gestational tissues and fetal fibronectin in cervico-vaginal fluids before the onset of labour at term. DESIGN: Cross-sectional, observational study. SAMPLES: Amnion, chorion laeve and decidua were collected from 24 term pregnant women following elective caesarean section. Samples of cervico-vaginal secretions were obtained from the same women immediately before caesarean section. METHODS: PGHS-1 and PGHS-2 mRNA levels in tissues were determined by specific ribonuclease protection assays. Fetal fibronectin concentrations in the cervico-vaginal fluids were measured by enzyme-linked immunosorbent assay. The abundance of PGHS mRNA was compared between groups of patients with the same mean gestational age but different cervico-vaginal fetal fibronectin levels. Linear regression analysis was used to determine the association between PGHS levels and fetal fibronectin. RESULTS: Two groups of women were identified who had significantly different fetal fibronectin values but the same gestational ages. The group with the higher fetal fibronectin concentrations had significantly higher PGHS- 1 and PGHS-2 mRNA levels in the chorion laeve and higher PGHS-2 mRNA levels in the amnion, than the group with lower fetal fibronectin concentrations. PGHS- 1 and PGHS-2 mRNA levels in the chorion laeve and PGHS-2 mRNA in the amnion showed an overall significant association with fetal fibronectin levels. CONCLUSIONS: High concentrations of fetal fibronectin in cervico-vaginal secretions before the onset of spontaneous labour at term are associated with high levels of PGHS-2 mRNA in the chorion laeve and the amnion and of PGHS- 1 mRNA in the chorion laeve. Increased expression of PGHS in these tissues may therefore be involved in the events leading to term birth.

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.

Prostaglandin endoperoxide H synthase mRNA expression in the human amnion and decidua during pregnancy and in the amnion at preterm labour.

We have examined the expression of prostaglandin endoperoxide H synthase (PGHS) isoenzymes in the amnion and the decidua during gestation, and the abundance of PGHS mRNA in the amnion at idiopathic preterm labour. PGHS-1 and -2 mRNA abundance in the amnion, determined with ribonuclease protection assays, was significantly (P< 0.05) higher at term than earlier during pregnancy. In contrast, neither PGHS-1 and -2 mRNA values, nor PGHS-specific activity, measured with a cell-free assay, was different in the decidua at term as compared to earlier gestational ages. In individual term patients, PGHS-2 mRNA values in the amnion were positively correlated with PGHS-2 mRNA values in the chorion laeve. PGHS-1 and -2 mRNA abundance was higher (P < 0.05) in the amnion after idiopathic preterm labour than in the absence of labour at the same gestational age (28-35 weeks). Thus, PGHS-1 and -2 are induced in the amnion at term. Furthermore, amniotic PGHS-2 changes in co-ordination with PGHS-2 concentrations in the chorion laeve. PGHS is not induced in the decidua at term. Increased amniotic PGHS expression may contribute to the enhanced intrauterine prostaglandin synthesis before term labour. Both PGHS isoenzymes may participate in the increase of PGHS activity in the amnion at preterm birth.

Regulation of prostaglandin H2 synthase-2 expression in primary human amnion cells by tyrosine kinase dependent mechanisms.

Prostaglandin H2 synthase (PGHS)-1 and PGHS-2 expression was examined in primary cultures of human amnion cells, an in vitro model of amnion tissue. Epidermal growth factor (EGF), the protein kinase C (PKC) activating phorbol ester TPA, and the protein phosphatase inhibitor, okadaic acid (OA), stimulated PGHS activity and the level of PGHS-2 mRNA, but did not affect the level of PGHS-1 mRNA. In situ hybridization suggested that the same population of cells responded to EGF, TPA and OA. Okadaic acid promoted PGHS activity independently of PKC. EGF stimulated the activity of extracellular signal-regulated protein kinase (Erk) and N-terminal c-Jun kinase (Jnk). OA increased Jnk activity but had no effect on Erk activity, while TPA had no influence on either Erk or Jnk activity. PD098059, a selective inhibitor of the Erk-activating kinase MEK, blocked the stimulation of PGHS expression by EGF, but did not decrease stimulation in response to OA. Herbimycin A, a tyrosine kinase inhibitor, suppressed the stimulation of PGHS activity and PGHS-2 mRNA abundance by all three stimulants, and blocked signalling via the Erk and Jnk mitogen-activated protein kinase pathways. Thus, growth factor stimulation, PKC activation and protein phosphatase inhibition induced the expression of PGHS-2 in primary amnion cells by distinct regulatory mechanisms involving tyrosine kinase(s). Tyrosine kinase inhibitors may constitute a new category of PGHS-2 inhibitors that act by blocking the expression of the enzyme.

Prostaglandin endoperoxide H synthase (PGHS) activity and PGHS-1 and -2 messenger ribonucleic acid abundance in human chorion throughout gestation and with preterm labor.

Term and preterm parturition is associated with elevated intrauterine PG production. Although an increase of PG synthesis by the fetal membranes during term labor is well documented, there is little data available regarding the prostanoid production of these tissues at term, before the spontaneous onset of labor. In the present study, we determined the expression of PG H2 synthase (PGHS), the committing and rate-limiting enzyme of prostanoid biosynthesis, in the chorion laeve during gestation. Tissues were collected from 18 patients at term (37-41 weeks of gestation) and from 13 patients between 17 and 35 weeks of pregnancy. None of the patients were in labor. PGHS-specific activity and the abundance of messenger RNAs (mRNAs) encoding the two PGHS isoenzymes (the constitutive PGHS-1 and the inducible PGHS-2) were measured by a cell-free enzyme assay and specific ribonuclease protection assays, respectively. PGHS-specific activity as well as PGHS-1 and -2 mRNA levels were significantly (P < 0.01) higher at term before labor than earlier during gestation. Furthermore, PGHS activity at term exhibited significant positive correlation with PGHS-2 mRNA levels, but not with PGHS-1 mRNA levels. In situ hybridization indicated that the expression of both PGHS mRNAs increased in the epithelial and the mesenchymal cells of the amnion and the chorion laeve at term. Additionally, PGHS activity and mRNA levels were determined in the chorion laeve of a group of patients who gave birth spontaneously before term (30.6 +/- 1 weeks, mean +/- SEM, n = 5), and the values were compared with a group who delivered by cesarean section before labor at a similar gestational age (31.9 +/- 1.4 weeks, n = 5, P > 0.05 vs. the preterm labor group). None of the patients exhibited signs of genital tract infection. PGHS-specific activity and PGHS-1 and -2 mRNA levels were significantly higher in the preterm labor group than in the group who delivered preterm without labor. In situ hybridization suggested that the enhanced PGHS-1 and -2 mRNA expression occurred predominantly in the mesenchymal cells of the fetal membranes at preterm labor. Thus, PGHS-1 and -2 expression increases in the chorion laeve at term before labor, with PGHS-2 as the functionally prevalent isoform. This supports the possibility that PGs originating in the fetal membranes promote the onset of normal labor. Furthermore, preterm labor is associated with the elevated expression of the two PGHS isoenzymes in the chorion laeve. The maturation of the fetal membranes in preparation for term labor involves both the epithelial and the mesenchymal cells, whereas preterm labor is accompanied by the maturation of the mesenchymal tissue components, as reflected by PGHS expression. This difference may have implications in the early recognition of preterm labor.

Prostaglandin endoperoxide H synthase-1 and -2 mRNA levels and enzyme activity in human decidua at term labor.

OBJECTIVE: To determine the labor-related changes of prostaglandin endoperoxide H synthase (PGHS) activity and PGHS-1 and -2 abundance in term decidua and to assess the contribution of the PGHS isoforms to the total PGHS activity present in the tissue. METHODS: Decidua was collected after elective cesarean delivery (CD) or spontaneous labor (SL) at term. Prostaglandin endoperoxide H synthase activity was determined in microsomal fractions, and PGHS-1 and -2 mRNA levels were measured by ribonuclease protection assays. Prostaglandin endoperoxide H synthase-1 and -2 mRNAs were localized in tissue sections by in situ hybridization. RESULTS: Prostaglandin endoperoxide H synthase specific activity in decidua microsomes at CD was 111 +/- 3 pg prostaglandin-E2/minute/microgram protein (mean +/- standard error, N = 10 patients), not different from enzyme activity measured after SL (110 +/- 27 N = 10 patients, P = .97, Wilcoxon's rank sum test). Prostaglandin endoperoxide H synthase-1 mRNA abundance in CD tissues was 0.283 +/- 0.047 relative densitometric units (mean +/- standard error, n = 26 patients), which did not change with labor (SL: 0.329 +/- 0.073, n = 20 patients, P = .68). Prostaglandin endoperoxide H synthase-2 mRNA abundance was also unaffected by labor (CD: 0.933 +/- 0.255, n = 27 patients; SL: 0.714 +/- 0.179, n = 23 patients, mean +/- standard error, P = .66). Prostaglandin endoperoxide H synthase specific activity was positively and significantly (P < .05) correlated with both PGHS-1 and -2 mRNA levels. In situ hybridization showed the pervasive presence of both PGHS mRNAs in decidua cells with no detectable changes associated with labor. CONCLUSION: Both isoforms of PGHS are present in term decidua and contribute to enzyme activity and prostaglandin production. Mechanisms regulating decidual prostanoid biosynthesis at labor do not involve changing the levels of expression of the two PGHS isoforms.

Prostaglandin-endoperoxide H synthase-2 expression and activity increases with term labor in human chorion.

We investigated the changes in prostaglandin-endoperoxide H synthase (PGHS) specific activity and the levels and distribution of PGHS-1 and PGHS-2 mRNA in chorion collected at term before the onset of labor (CS) and after term labor and delivery (SL). PGHS specific activity and PGHS-2 mRNA abundance were higher in chorion collected after SL compared with that obtained at CS (P < 0.001); there was no difference in the levels of PGHS-1 mRNA between CS and SI, tissues. The increase in PGHS specific activity at SL was significantly correlated with PGHS-2 mRNA expression (P < 0.05) but not with PGHS-1 mRNA levels. In situ hybridization indicated that the pervasiveness of staining for PGHS-1 mRNA throughout full-thickness membranes did not change with labor onset; however, a greater number of cells expressed PGHS-2 in SL tissues. Our results demonstrate a selective increase in PGHS-2 expression and activity in chorion trophoblasts and mesenchymal cells with term labor onset. These observations are similar to those concerning amnion and imply that a concerted mechanism may exist in the fetal membranes to induce PGHS-2 expression at labor.

Regulation of prostaglandin endoperoxide H2 synthase in term human gestational tissues.

Increased production of prostaglandins by the gestational tissues is pivotal for the initiation and maintenance of human labour. A major source of prostaglandins in the pregnant human uterus is the amnion membrane, which synthesizes increased amounts of prostaglandin E2 (PGE2) at parturition. We have found that the activity of prostaglandin endoperoxide H2 synthase (PGHS), the enzyme catalyzing the committing step of prostanoid biosynthesis, increases significantly in the amnion at term and preterm labour, and also prior to the onset of clinical labour at term. Furthermore, the abundance of the mRNA encoding the inducible PGHS-2 isoenzyme was higher in the amnion after spontaneous delivery that before labour. The level of the constitutive PGHS-1 mRNA remained unchanged. In addition, we found a significant positive correlation between PGHS activity and the level of PGHS-2 mRNA, but not of PGHS-1 mRNA, in the individual tissue samples, also indicating that PGHS-2 was selectively induced in the amnion membrane at labour. The regulation of PGHS expression by agonists was studied using primary cultures of amnion cells. Glucocorticoid treatment enhanced the activity of PGHS and the level of PGHS-2 mRNA in the cultured cells, without affecting PGHS-1 mRNA abundance. The stimulation was glucocorticoid specific and was blocked by the glucocorticoid receptor antagonist RU486, suggesting that it was mediated by the glucocorticoid receptor. Inhibition of protein synthesis did not block the accumulation of PGHS-2 mRNA showing that the steroid acted directly, without inducing an intervening protein. Protein kinase C activator and protein phosphatase inhibitor compounds and epidermal growth factor also promoted PGHS-2 mRNA expression, demonstrating the involvement of protein kinase dependent mechanisms in PGHS-2 regulation. However, the role of these effectors in the in vivo control of PGHS-2 expression remains to be determined.

Glucocorticoids stimulate the expression of prostaglandin endoperoxide H synthase-2 in amnion cells.

Corticosteroids increase the production of prostaglandin E2 (PGE2) and the activity of prostaglandin endoperoxide H synthase (PGHS) in cultured amnion cells, although they inhibit prostanoid biosynthesis in numerous other cell types. This suggests that glucocorticoids control the level of PGHS in amnion cells by a hitherto unexplored, positive regulatory mechanism. We have tested the possibility that corticosteroids act by stimulating the expression of messenger RNAs (mRNAs) encoding one or both isoforms of PGHS. Ribonuclease protection assays were used to determine the levels of PGHS-1 and -2 mRNAs and, for reference, gamma-actin mRNA levels in confluent primary cultures of human amnion cells. In untreated cultures, PGHS-1 and -2 mRNA levels were low, often not reaching the level of detection. Dexamethasone (DEX) treatment for 4 h resulted in a measurable level of PGHS-2 mRNA, which increased further 10-fold and 20-fold after incubation with the glucocorticoid for 8 h and 16 h, respectively. The stimulation was dependent on DEX concentration, and was concomitant with an increase in the capacity of the cells to metabolize arachidonic acid to PGE2. PGHS-1 mRNA levels remained low in DEX-treated cells, while the gamma-actin message level showed no change. Estradiol and progesterone had no influence on PGHS-2 mRNA expression, but cortisol increased the PGHS-2 mRNA abundance. The glucocorticoid antagonist RU486 blocked the effect of DEX. Conditioned media of DEX-treated cells did not contain steroid-induced factor(s) stimulating PGE2 production. Inhibition of protein synthesis by cycloheximide potentiated the effect of DEX, and raised the abundance of PGHS-1, PGHS-2, and gamma-actin mRNAs in untreated cells. DEX did not affect the stability of the PGHS-2 mRNA. These results show that glucocorticoids promote PGE2 synthesis by amnion cells by stimulating the expression of PGHS-2 mRNA in a receptor-dependent, selective, and immediate fashion.

Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset.

Increased prostaglandin (PG) production within the uterine compartment has a pivotal role in the processes leading to labor onset in women. Two PG endoperoxide-H synthase (PGHS) isoenzymes have been identified in a number of cell types. PGHS-1 is constitutively expressed in most cases, whereas PGHS-2 expression is rapidly induced by several agonists. The aims of this study were to determine the levels of PGHS-1 and PGHS-2 expression before and after spontaneous labor (SL) onset in the amnion and to assess the contribution of PGHS-1 and PGHS-2 to enzyme activity. We established and validated ribonuclease protection assays to quantify PGHS-1 and PGHS-2 messenger ribonucleic acid (mRNA) levels in the amnion. PGHS enzyme activity was measured with an established assay. The antisense RNA probes used in the protection assays were generated using human PGHS-1 and PGHS-2 complementary DNAs. These probes specifically detected the 2.8-kilobase mRNA of PGHS-1 and the 4.8-kilobase mRNA of PGHS-2 in amnion RNA samples on Northern blots. We measured mRNA levels in amnion from patients after SL at term and from patients not in labor undergoing elective cesarean section (CS) at term. PGHS-2 mRNA levels were markedly higher after SL compared to levels in CS amnion [5.18 +/- 1.08 (n = 16) and 2.27 +/- 0.50 (n = 15), densitometric units, respectively; P < 0.02], whereas there was no difference in PGHS-1 mRNA levels after labor compared with CS samples. PGHS-2 mRNA levels were also positively correlated with PGHS enzyme activity in 4 separate assays with a total of 25 patients (r = 0.65-0.88; P < 0.05). There was no correlation between PGHS-1 mRNA levels and enzyme activity. We conclude that PGHS-2 mRNA is present in human amnion; its levels are elevated after SL onset, and they are correlated with enzyme activity. The stimulation of PGHS activity at labor onset probably involves increased expression of PGHS-2. The expression of PGHS-1 does not change in association with labor in human amnion.

Studies on glucocorticoid hormone actions in the regulation of human amnion PGHS.

The human amnion is an excellent model in which to study the regulation of the expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNA by glucocorticoids, as glucocorticoids can both stimulate and inhibit PGHS activity and can have their stimulatory effect reversed to an inhibitory one. Our studies suggest the presence of a glucocorticoid-induced inhibitory protein which affects the post-transcriptional activity of PGHS-2 mRNA. Glucocorticoid-induced expression of PGHS-2 mRNA may be dependent on protein phosphorylation.