Prostaglandin H Synthase-2 mRNA Research Articles

Prostaglandins mediate the fetal pulmonary response to intrauterine inflammation

Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1β and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.

Low-Intensity Pulsed Ultrasound Modulates Shear Stress Induced PGHS-2 Expression and PGE2 Synthesis in MLO-Y4 Osteocyte-Like Cells

Fluid shear stress (SS) has been shown to be a prevailing physiological stimulus in the regulation of bone cell metabolism and so are the exogenous biomechanical forces, like ultrasound (US) and vibration. The purpose of this study is to elaborate the interplay of laminar fluid SS with low-intensity pulsed US in the regulation of prostaglandin H synthase 2 (PGHS-2) and prostaglandin E2 (PGE2). Murine long bone osteocyte-like (MLO-Y4) cells were exposed to various regimes of US (1.5 Hz, 30 mW/cm2) and SS (19 dyn/cm2) alone and sequentially. Changes in PGHS-2 gene expression levels were quantified at 3 and 24 h using real-time RT-PCR. PGE2 levels in the culture media were measured using enzyme immunoassay at 3 and 24 h. PGE2 levels significantly increased after exposure to SS for 3 and 24 h by 2.17±0.02 and 5.47±0.42-fold, respectively, compared to control cells. A 20 min US treatment prior to SS significantly increased SS PGE2 levels 2.95±0.18 and 2.90±0.50-fold at 3 and 24 h, respectively. US also significantly increased PGHS-2 mRNA levels in cells exposed to SS. SS caused a 2.74 ± 0.49-fold increase in PGHS-2 mRNA levels at 3 h and a significant 3.70±0.25-fold increase at 24 h relative to control. A 20 min US treatment caused 1.35±0.49 and 2.44±0.82-fold increase in PGHS-2 mRNA levels in cells exposed to SS at 3 and 24 h, respectively. These results indicate that combining US with SS may have a more anabolic benefit for bone tissue than either stimulus alone.

Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy

The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n = 4), late diestrus (LD, n = 4), early luteolysis (EL, n = 4) and after luteolysis (AL, n = 4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n = 4), 15 (P15, n = 4), 18 (P18, n = 4) and 22 (P22; n = 4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test ( α = 0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early pregnancy along with PTGER2 while PTGFR expression was suppressed. These findings suggest that possible luteotrophic action of PGE 2 is required in early equine pregnancy. PTGS1 is only upregulated later in the early pregnancy suggesting that it is not involved in luteolysis, but could be the main PTGS enzyme at this time during early pregnancy. An increase in HPGD and SLCO2A1 levels on P22 indicates a tight regulation of PTG action by pregnancy.

Stretch and interleukin 1 beta: Pro‐labour factors with similar mitogen‐activated protein kinase effects but differential patterns of transcription factor activation and gene expression

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

The Mitogen-Activated Protein Kinase Dependent Expression of Prostaglandin H Synthase-2 and Interleukin-8 Messenger Ribonucleic Acid by Myometrial Cells: The Differential Effect of Stretch and Interleukin-1β

Infection and uterine stretch are the common causes of preterm labor. IL-1beta plays a key role in infection-induced preterm labor and increases prostaglandin H synthase 2 (PGHS-2) and IL-8 expression. We have shown that mechanical stretch of uterine myocytes in vitro up-regulates the expression of PGHS-2 and IL-8. In this study, we tested the hypotheses that both IL-1beta and mechanical stretch increase the myometrial expression of PGHS-2 and IL-8 via MAPK activation and that their effects are synergistic. MAPK activation was assessed in myocytes obtained from pregnant women undergoing cesarean section before the onset of labor after exposure to IL-1beta and stretch either alone or in combination. Specific inhibitors of ERK, p38, and c-Jun N-terminal kinase were used to define the role of each in the increased expression of PGHS-2 and IL-8 mRNA. We found that both IL-1beta and stretch activated all three MAPK subtypes but that they had no synergistic effect. The inhibitor studies showed that stretch-induced increases in both PGHS-2 and IL-8 mRNA expression were ERK1/2 and p38 dependent and that IL-1beta-induced increases of PGHS-2 mRNA expression were also ERK1/2 and p38 dependent, but those of IL-8 were dependent only on ERK1/2 activation. These data show that exposure of human uterine myocytes to both stretch and IL-1beta activates the MAPK system, which is responsible for the increase in PGHS-2 and IL-8 mRNA expression. We found no evidence of a synergistic effect of IL-1beta and stretch on myometrial expression of PGHS-2 and IL-8 mRNA.

Induction of immunoreactive prostaglandin H synthases 1 and 2 and fos in response to cerebral hypoperfusion in late-gestation fetal sheep

The goal of the present study was to localize and quantify immunoreactive prostaglandin H synthase 1 (PGHS-1), PGHS-2, and Fos expression by immunohistochemistry in the fetal brain 30 minutes and 2 hours after the onset of a 10-minute period of cerebral hypoperfusion in barodenervated and chemodenervated fetal sheep. Fetal sheep of known gestational age were studied intact or sinoaortic denervated. Fetuses were sacrificed and tissues recovered for immunohistochemistry or real-time polymerase chain reaction measurements of protein and mRNA, respectively. Some fetuses were subjected to brachiocephalic occlusion, produced by inflation of an extravascular balloon occluder around the brachiocephalic artery. Immunohistochemistry results were quantified using image analysis, and mRNA was quantified by estimation of cycle threshold in generation of PGHS-1 or PGHS-2 amplicons. Sinoaortic denervation by itself did not alter the abundance of PGHS-1 or PGHS-2 protein in any brain region, although the denervation did reduce the abundance of PGHS-1 mRNA in hypothalamus. We assessed PGHS-1, PGHS-2, and Fos immunoreactive protein abundance by image analysis of histologic sections stained for the respective proteins using immunohistochemistry. Cerebral hypoperfusion increased the intensity of staining of immunoreactive PGHS-1, PGHS-2, and Fos in the anterior pituitary, hippocampus, and cerebellum. In the cerebral microvasculature, the intensity of PGHS-1 and Fos was significantly greater, and in the cerebral cortex, the intensity of PGHS-2 was significantly greater. Changes in the amount of immunostaining in the nucleus of tractus solitarius and paraventricular nucleus were not statistically significant. Cerebral hypoperfusion altered the expression and distribution of prostaglandin biosynthetic enzymes in ovine fetal brain by a mechanism that is independent of baroreceptor and chemoreceptor afferent activity.

The in Vivo Control of Prostaglandin H Synthase-2 Messenger Ribonucleic Acid Expression in the Human Amnion at Parturition

Prostaglandin H synthase-2 (PGHS-2) activity and mRNA rise in the human amnion at late gestation, contributing to the increase in intrauterine PG production crucial for labor and delivery. In the present investigation we have determined the mechanism that controls amniotic PGHS-2 mRNA levels in vivo at term parturition. Amnion membranes were collected after elective cesarean section (n = 20), and after spontaneous labor (n = 20). PGHS-2 relative gene transcription rates were determined by transcriptional run-on, and PGHS-2 mRNA and heterogeneous nuclear RNA (hnRNA) relative abundance were measured by quantitative real-time RT-PCR. The PGHS-2 mRNA degradation rate was determined by incubating amnion in the presence of the transcription inhibitor 5,6-dichlorobenzimidazole riboside. The dynamics of PGHS-2 hnRNA and mRNA abundance were characterized in 0- to 24-h tissue incubations. The PGHS-2 relative gene transcription rate was a significant (P < 0.05) predictor of PGHS-2 hnRNA and mRNA abundance, and PGHS-2 hnRNA was also a predictor (P < 0.01) of PGHS-2 mRNA levels both before and after labor. Interestingly, even though PGHS-2 gene activity remained unchanged, PGHS-2 mRNA abundance increased with labor and displayed constitutive stability before and after labor. PGHS-2 mRNA levels spontaneously increased by 400% (P < 0.01) upon incubation for 24 h, whereas the transcription rate dropped by 95% during the first 2 h, then rebounded significantly between 6–24 h. Thus, PGHS-2 mRNA abundance is transcriptionally controlled in term amnion. Labor does not increase PGHS-2 gene activity or mRNA stability. The PGHS-2 gene is probably induced before labor by a factor(s) originating in the amnion membrane, and the resulting stable mRNA accumulates progressively in the tissue throughout labor and delivery.

The in vivo control of prostaglandin H synthase-2 messenger ribonucleic acid expression in the human amnion at parturition.

Prostaglandin H synthase-2 (PGHS-2) activity and mRNA rise in the human amnion at late gestation, contributing to the increase in intrauterine PG production crucial for labor and delivery. In the present investigation we have determined the mechanism that controls amniotic PGHS-2 mRNA levels in vivo at term parturition. Amnion membranes were collected after elective cesarean section (n = 20), and after spontaneous labor (n = 20). PGHS-2 relative gene transcription rates were determined by transcriptional run-on, and PGHS-2 mRNA and heterogeneous nuclear RNA (hnRNA) relative abundance were measured by quantitative real-time RT-PCR. The PGHS-2 mRNA degradation rate was determined by incubating amnion in the presence of the transcription inhibitor 5,6-dichlorobenzimidazole riboside. The dynamics of PGHS-2 hnRNA and mRNA abundance were characterized in 0- to 24-h tissue incubations. The PGHS-2 relative gene transcription rate was a significant (P < 0.05) predictor of PGHS-2 hnRNA and mRNA abundance, and PGHS-2 hnRNA was also a predictor (P < 0.01) of PGHS-2 mRNA levels both before and after labor. Interestingly, even though PGHS-2 gene activity remained unchanged, PGHS-2 mRNA abundance increased with labor and displayed constitutive stability before and after labor. PGHS-2 mRNA levels spontaneously increased by 400% (P < 0.01) upon incubation for 24 h, whereas the transcription rate dropped by 95% during the first 2 h, then rebounded significantly between 6-24 h. Thus, PGHS-2 mRNA abundance is transcriptionally controlled in term amnion. Labor does not increase PGHS-2 gene activity or mRNA stability. The PGHS-2 gene is probably induced before labor by a factor(s) originating in the amnion membrane, and the resulting stable mRNA accumulates progressively in the tissue throughout labor and delivery.

Regulation of prostaglandin availability in human fetal lung by differential localisation of prostaglandin H synthase-1 and prostaglandin dehydrogenase.

Specialisation of the respiratory portion of human fetal lung commences around 20-24 weeks gestation. In contrast, human fetal lung in vitro has the capacity to self-differentiate from 12 weeks gestation when grown in media devoid of growth factors or hormones, suggesting activation of autocrine or paracrine factors in vitro, or removal of the fetus from in utero inhibitory mechanisms. Prostaglandins play a key role during in vitro human fetal lung development and are synthesised by prostaglandin H synthase-1 (PGHS-1) and inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH) with formation of inactive 13,14-dihydro-15-keto-prostaglandins. We have used quantitative immunohistochemistry to determine expression and localisation of PGHS-1, PGDH, PGE2 and 13,14-dihydro-15-keto-PGE2 (PGEM) in human fetal lung with in situ hybridisation to localise PGHS-1 and PGDH mRNA. For the catabolic enzyme PGDH, amounts of mRNA, protein and enzyme product PGEM are increased within epithelium of distal as compared to more proximal airways. For PGHS-1, comparable amounts of mRNA, protein and enzyme product PGE2 are found in proximal and distal lung epithelium. Catabolism by PGDH is a sensitive mechanism for regulating bioavailability of prostaglandins and we propose that active catabolism of prostaglandins within human fetal lung epithelium is an inhibitory mechanism retarding epithelial differentiation in utero.

Expression of prostaglandin H synthase-2 in human brain tumors.

Numerous studies have demonstrated that prostaglandin H synthase-2 (PHS-2) is involved in gastrointestinal carcinogenesis, and that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PHS, can reduce the risk of colon cancer. In brain tumors, elevated prostaglandin production and its correlation to anaplastic grade of gliomas have been demonstrated. To determine whether the increased prostaglandin production is due to enhanced expression of PHS-2 and whether the up-regulation of PHS-2 has any correlation to histopathological findings in brain tumors, we evaluated the profile of PHS expression in several human glioma cell lines and surgical specimens from patients with various types of brain tumors. In glioma cell lines, five out of six cell lines showed constitutive expression of PHS-2, whereas PHS-1 was weakly expressed in all of them. All surgical specimens, except an ependymoma, which expressed both isozymes equally, expressed PHS-2 mRNA predominantly. Immunohistochemistry of various types of brain tumors, including six glioblastomas, nine astrocytomas, six meningiomas, five medulloblastomas, four craniopharyngiomas, three ependymomas, three neurinomas, two oligodendrogliomas, two malignant lymphomas, two dysembryoplastic neuroepitherial tumors and one metastatic brain tumor showed PHS-2 staining in most cases. In gliomas, astrocytomas (grade 2 and 3) were strongly stained, but the staining intensity of glioblastomas was relatively weak. Meningiomas and a metastatic brain tumor were also strongly stained. Our data thus suggest that most brain tumors express PHS-2, which may also play a role in tumorigenesis in the brain.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

Differential expression of myometrial oxytocin receptor and prostaglandin H synthase 2, but not estrogen receptor alpha and heat shock protein 90 messenger ribonucleic acid in the gravid horn and nongravid horn in sheep during betamethasone-induced labor.

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor alpha (ERalpha), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130-140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101-110 dGA (n = 3), 111-120 dGA (n = 3), 121-130 dGA (n = 3), 131-140 dGA (n = 3), and 141-145 dGA (n = 4) and from ewes in spontaneous term labor (n = 4). All ewes were carrying single fetuses. Myometrium was also collected from ovariectomized nonpregnant ewes treated with saline (n = 5), estradiol (50 microg/day; n = 5), progesterone (0.3 g, intravaginally; n = 5), and estradiol plus progesterone (n = 5). Myometrial RNA was extracted and analyzed by Northern blot for OTR, PGHS2, ERalpha, and Hsp90 mRNA, normalized for 18S ribosomal RNA or beta-actin. ERalpha, Hsp90, OTR, and PGHS2 mRNA were all significantly up-regulated during betamethasone-induced labor (P < 0.01) in gravid and nongravid horn myometrium. The level of gravid horn OTR mRNA during labor was 3 times the level of nongravid horn OTR mRNA (P < 0.0001). Gravid horn PGHS2 mRNA was also higher than nongravid horn PGHS2 (P < 0.02). In contrast, in spontaneous term labor nongravid horn, ERalpha and Hsp90 mRNA were similar to gravid horn. Myometrial ERalpha and Hsp90 mRNA remained unchanged throughout late pregnancy and increased at spontaneous term labor (P < 0.05). In contrast, myometrial OTR increased around 130 dGA (P < 0.01) and further increased at spontaneous term labor (P < 0.02). Progesterone significantly inhibited myometrial OTR mRNA expression in nonpregnant sheep and estradiol antagonized progesterone's inhibitory effect. Mechanical stretch differentially regulated mCAP mRNA expression in the ovine gravid horn and nongravid horn. Mechanical stretch appears largely responsible for increased OTR mRNA and to a lesser degree PGHS2 mRNA. In addition, endocrine factors may be required for full activation of OTR and PGHS2 mRNA associated with labor. ERalpha and Hsp90 mRNA are not under the control of uterine stretch in keeping with our previous results, indicating that systemic hormones such as estradiol, are prime regulators for these two mCAP mRNA expression during labor.

Differential Expression of Myometrial Oxytocin Receptor and Prostaglandin H Synthase 2, But Not Estrogen Receptor and Heat Shock Protein 90 Messenger Ribonucleic Acid in the Gravid Horn and Nongravid Horn in Sheep during Betamethasone-Induced Labor

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor α (ERα), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130–140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101–110 dGA (n = 3), 111–120 dGA (n = 3), 121–130 dGA (n = 3), 131–140 dGA (n = 3), and 141–145 dGA (n = 4) and from ewes in spontaneous term labor (n = 4). All ewes were carrying single fetuses. Myometrium was also collected from ovariectomized nonpregnant ewes treated with saline (n = 5), estradiol (50 μg/day; n = 5), progesterone (0.3 g, intravaginally; n = 5), and estradiol plus progesterone (n = 5). Myometrial RNA was extracted and analyzed by Northern blot for OTR, PGHS2, ERα, and Hsp90 mRNA, normalized for 18S ribosomal RNA orβ -actin. ERα, Hsp90, OTR, and PGHS2 mRNA were all significantly up-regulated during betamethasone-induced labor (P< 0.01) in gravid and nongravid horn myometrium. The level of gravid horn OTR mRNA during labor was 3 times the level of nongravid horn OTR mRNA (P < 0.0001). Gravid horn PGHS2 mRNA was also higher than nongravid horn PGHS2 (P < 0.02). In contrast, in spontaneous term labor nongravid horn, ERα and Hsp90 mRNA were similar to gravid horn. Myometrial ERα and Hsp90 mRNA remained unchanged throughout late pregnancy and increased at spontaneous term labor (P < 0.05). In contrast, myometrial OTR increased around 130 dGA (P < 0.01) and further increased at spontaneous term labor (P< 0.02). Progesterone significantly inhibited myometrial OTR mRNA expression in nonpregnant sheep and estradiol antagonized progesterone’s inhibitory effect. Mechanical stretch differentially regulated mCAP mRNA expression in the ovine gravid horn and nongravid horn. Mechanical stretch appears largely responsible for increased OTR mRNA and to a lesser degree PGHS2 mRNA. In addition, endocrine factors may be required for full activation of OTR and PGHS2 mRNA associated with labor. ERα and Hsp90 mRNA are not under the control of uterine stretch in keeping with our previous results, indicating that systemic hormones such as estradiol, are prime regulators for these two mCAP mRNA expression during labor.

Progesterone and Prostaglandin H Synthase‐2 Involvement in Alcohol‐Induced Preterm Birth in Mice

Recently, an association between alcohol consumption during pregnancy and shortened gestational length has been reported, but the underlying mechanisms remain unknown. Progesterone (P4) and prostaglandins have been shown to play important roles in parturition in both human and animal models. Recently, it has been suggested that prostaglandin H synthase-2 (PGHS-2) is responsible for prostaglandin changes associated with term and preterm labor. It is possible that alcohol induces preterm birth by altering P4 or PGHS-2 levels. These studies were designed to determine the role of P4 and PGHS-2 in alcohol-induced preterm labor in mice. Experiment 1: Pregnant dams treated with either vehicle or alcohol (6 g/kg, intragastrically) on gestational day (GD) 16 were killed at various times in gestation up to the time of delivery. Plasma P4 levels were measured by radioimmunoassay and uterine PGHS-2 mRNA expression was measured by Ribonuclease Protection Assay. Results indicated that alcohol treatment was associated with an earlier decline in plasma P4 levels and an earlier rise in uterine PGHS-2 mRNA levels during gestation. Experiment 2: Pregnant C57BL/6J females were treated with either P4 (2.0 mg, subcutaneously) or vehicle (sesame oil) 2 hr before receiving either 6 g/kg alcohol (intragastrically) or vehicle (isocaloric sucrose) on gestational day (GD) 16. Results indicate that P4 pretreatment effectively antagonized alcohol-induced preterm delivery. Experiment 3: On GD16, pregnant dams received either 100 mg/kg nimesulide (a specific PGHS-2 inhibitor) or vehicle (saline) subcutaneously, 2 hr before treatment with either 6 g/kg alcohol (given intragastrically) or isocaloric sucrose. Nimesulide was effective in antagonizing alcohol-induced preterm labor. Together, these data suggest that both P4 and PGHS-2 may play roles in alcohol-induced preterm birth.

Progesterone and Prostaglandin H Synthase-2 Involvement in Alcohol-Induced Preterm Birth in Mice

Background : Recently, an association between alcohol consumption during pregnancy and shortened gestational length has been reported, but the underlying mechanisms remain unknown. Progesterone (P4) and prostaglandins have been shown to play important roles in parturition in both human and animal models. Recently, it has been suggested that prostaglandin H synthase-2 (PGHS-2) is responsible for prostaglandin changes associated with term and preterm labor. It is possible that alcohol induces preterm birth by altering P4 or PGHS-2 levels. These studies were designed to determine the role of P4 and PGHS-2 in alcohol-induced preterm labor in mice. Methods : Experiment 1: Pregnant dams treated with either vehicle or alcohol (6 g/kg, intragastrically) on gestational day (GD) 16 were killed at various times in gestation up to the time of delivery. Plasma P4 levels were measured by radioimmunoassay and uterine PGHS-2 mRNA expression was measured by Ribonuclease Protection Assay. Results indicated that alcohol treatment was associated with an earlier decline in plasma P4 levels and an earlier rise in uterine PGHS-2 mRNA levels during gestation. Experiment 2: Pregnant C57BW6J females were treated with either P4 (2.0 mg, subcutaneously) or vehicle (sesame oil) 2 hr before receiving either 6 g/kg alcohol (intragastrically) or vehicle (isocaloric sucrose) on gestational day (GD) 16. Results indicate that P, pretreatment effectively antagonized alcohol-induced preterm delivery. Experiment 3: On GD16, pregnant dams received either 100 mgkg nimesulide (a specific PGHS-2 inhibitor) or vehiclc (saline) subcutaneously, 2 hr before treatment with either 6 g/kg alcohol (given intragastrically) or isocaloric sucrose. Nimesulide was effective in antagonizing alcohol-induced preterm labor. Conclusions : Together, these data suggest that both P4 and PGHS-2 may play roles in alcohol-induced preterm birth.

Cytokines regulate prostaglandin H synthase-1 transcription in human amnion-derived cells

We have isolated the prostaglandin H synthase-1 (PGHS-1) promoter from the human amnion cell line WISH by long template PCR. The fragment was 1124 base pairs in length and shared a 96% sequence identity with the sequence in GenBank. The putative transcription start site is located 18 bp upstream of the start codon. The sequence is TATA-less, but contains multiple Sp-1 sites and a GC box at −132. The fragment was subcloned into the promoterless reporter construct pBLCAT3 to produce the promoter reporter construct pPGHS1CAT. pPGHS1CAT expression in amnion-derived AV3 cells was inhibited by the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β). PGHS-1 mRNA levels however, were unchanged over a 16-h time course with either treatment. These results suggest that PGHS-1 transcription is regulated in a negative manner by cytokines in human amnion-derived cells.

Effect of Taxol and Taxotere on Gene Expression in Macrophages: Induction of the Prostaglandin H Synthase-2 Isoenzyme

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-gamma increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-gamma altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.

Effects of ischemia on prostaglandin H synthase-2 expression in piglet choroid plexus

We examined effects of ischemia on expression of prostaglandin H synthase-1 (PGHS-1) and prostaglandin H synthase-2 (PGHS-2) in piglet choroid plexus. Ten minutes of ischemia was induced by increasing intracranial pressure. Whole choroid plexus was removed and fixed and/or frozen after 1, 2, 4, and 8 h of recovery from anoxic stress. In addition, tissues were obtained from untreated animals or from time control animals. Tissues were analyzed for mRNA, using RNase protection assays, and for proteins, using immunohistochemical approaches. Limited, but detectable PGHS-2 immunoreactivity was present in choroid plexus under normal conditions, and there was no difference between time-control and non-treated animals. Further, PGHS-2 mRNA increased by 2–4 h after ischemia, and enhanced immunoreactivity for PGHS-2 was present at 8 h after ischemia. Enhanced immunoreactivity for PGHS-2 was present in vascular endothelial cells as well as cuboidal epithelial cells and macrophages. In contrast, PGHS-1 mRNA did not increase following ischemia. We conclude that PGHS-2 is present in piglet choroid plexus under normal conditions and that ischemia increases levels of PGHS-2 in choroid plexus.

Effects of anoxic stress on prostaglandin H synthase isoforms in piglet brain

We examined effects of ischemia and asphyxia on levels of prostaglandin H synthase-1 (PGHS-1) and prostaglandin H synthase-2 (PGHS-2) in piglet brain. Ischemia was induced by increasing intracranial pressure and asphyxia was induced by turning off the respirator. Duration of anoxic stress was 10 min. In some animals, indomethacin (5 mg/kg, i.v.) or 7-nitroindazole (7-NI) was administered prior to ischemia to block PGHS or brain nitric oxide synthase (bNOS), respectively. Tissues from cerebral cortex and hippocampus were removed and fixed and/or frozen after 1, 2, 4 and 8 h of recovery from anoxic stress. In addition, tissues were obtained from untreated animals or from time control animals. Levels of mRNA and proteins were determined using RNase protection assay and immunohistochemical approaches, respectively. In the tissues studied, only a few neurons were immunopositive for PGHS-1, and neither ischemia or asphyxia affected PGHS-1 immunostaining at 8 h after recovery. Likewise, PGHS-1 mRNA did not increase following anoxic stress. In contrast, substantial PGHS-2 immunoreactivity was present in neurons and glial cells in the cerebral cortex and hippocampus and there was no difference between time control and non treated animals. PGHS-2 mRNA increased by 2–4 h after ischemia, and heightened immunoreactivity for PGHS-2 was present at 8 h after ischemia in cerebral cortex and hippocampus. However, asphyxia did not increase PGHS-2 mRNA or immunostaining. Indomethacin pretreatment inhibited increases in mRNA and protein for PGHS-2 after ischemia, while 7-NI had little effect on increases in PGHS-2 immunoreactivity. We conclude that: (1) PGHS-2 is the predominant isoform present in piglet cerebral cortex and hippocampus; (2) Ischemia but not asphyxia increases levels of PGHS-2; (3) Ischemia does not increase levels of PGHS-1; and (4) Indomethacin but not 7-NI attenuates ischemia-induced increases in PGHS-2.

Suppression of Prostaglandin H Synthase-2 Induction in Human Monocytes byin Vitroorin VivoAdministration of Interleukin 4

IL-4 is a potent modulator of monocyte function. Our previous studies demonstrated that the suppression of monocyte matrix metalloproteinase production by IL-4 is a result of its inhibition of PGE2synthesis, which was attributed to an effect on prostaglandin synthase. Here we report on thein vitroandin vivoeffects of IL-4 on monocyte prostaglandin H synthase-2 (PGHS-2) and its regulation by second messengers. Stimulation of monocytes with either LPS or Con A resulted in the induction of PGHS-2 which was significantly inhibited by IL-4. Inhibition of PGHS-2 mRNA and protein was detected at 0.05 to 0.1 ng/ml of IL-4 with substantial suppression at 10 to 20 ng/ml. If added later than 2 hr after LPS, IL-4 failed to suppress PGHS-2, indicating that IL-4 acted early in the signaling cascade. Moreover, the ability of exogenously added PGE2or Bt2cAMP to restore PGHS-2 production in IL-4-treated monocytes further suggested early disruption of the pathway. The early event inhibited by IL-4 did not involve suppression of phospholipase activity, because LPS-induced arachidonic acid release was relatively unaffected by IL-4. Unlike PGHS-2, PGHS-1, the constitutively expressed PGHS, was not modulated by IL-4. Thus, IL-4 appears to selectively block PGHS-2 synthesis, thereby blocking subsequent steps in the pathway leading to the production of matrix metalloproteinases. In an extension of these findings, we examined peripheral blood monocytes from cancer patients undergoing IL-4 therapy. In these cells the induction of PGHS-2 expression by LPS was significantly reduced compared to that of monocytes obtained prior to IL-4 therapy. Although perhaps not relevant as an antitumor mechanism, these findings have important implications in defining the potent anti-inflammatory activities of IL-4in vitroandin vivo.