Prostaglandin-endoperoxide Synthase 2 mRNA Research Articles

Nrf2-Mediated Ferroptosis Inhibition Exerts a Protective Effect on Acute-on-Chronic Liver Failure.

Although massive hepatocyte cell death and oxidative stress constitute major events of acute-on-chronic liver failure (ACLF), the relationship of ferroptosis with ACLF has yet to be explored. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of ferroptosis. However, if Nrf2 modulates ACLF through ferroptosis remains unknown. Here, the liver tissues of ACLF patients were collected and murine models of ACLF using carbon tetrachloride, D-galactosamine, and lipopolysaccharide as well as an H2O2-induced hepatocyte injury model were established. Upon ACLF, livers exhibited key features of ferroptosis, including lipid peroxidation (increase in malondialdehyde whereas a decrease in glutathione and nicotinamide adenine dinucleotide phosphate), and increased mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2). Ferroptosis inducer RSL-3 treatment aggravated liver damage, while ferroptosis inhibitor Ferrostatin-1 administration alleviated ACLF severity, manifesting with improved liver histopathological lesions and reduced serum ALT and AST. Compared with normal liver tissue, Nrf2 was upregulated in ACLF patients and murine models. Pharmacological activation of Nrf2 (Bardoxolone Methyl) attenuated liver damage, prevented lipid peroxidation, upregulated PTGS2 mRNA expression, and improved ferroptosis-specific mitochondrial morphology in vivo. In contrast, Nrf2 inhibitor ML385 exacerbated lipid peroxidation and liver injury. Collectively, Nrf2 plays a protective role in ACLF progression through repressing ferroptosis, which provides promising therapeutic cues for ACLF.

Carboxymethylated pachyman induces ferroptosis in ovarian cancer by suppressing NRF1/HO-1 signaling.

Carboxymethylated pachyman (CMP) is characterized by immune regulatory, antitumor and antioxidant activities. However, whether CMP contributes to the treatment of ovarian cancer has yet to be explored. The role of CMP in ovarian cancer cell death was analyzed using CCK-8 and flow cytometry assays. The data showed that CMP induced ovarian cancer cell death in a dose-dependent manner. Furthermore, CMP-induced cell death could be largely reversed by preincubation with ferrostatin-1 (Fer-1) but not 3-methyladenine or necrostatin-1. Reverse transcription-quantitative PCR analysis indicated that CMP significantly increased prostaglandin-endoperoxide synthase 2 (PTGS2) and Chac glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) mRNA levels, but preincubation with Fer-1 obviously reduced PTGS2 and CHAC1 mRNA levels in SKOV3 and Hey cells. The intracellular levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and Fe2+ were then quantified The data showed that 100 and 200 µg/ml CMP enhanced the production of SOD, MDA and Fe2+ but decreased GSH levels in SKOV3 and HEY cells. These data indicated that CMP could induce ferroptosis in ovarian cancer cells. More importantly, in vitro and in vivo studies indicated that CMP significantly suppressed nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), cystine/glutamate antiporter system X(c)(−) (xCT) and glutathione peroxidase 4 (GPX4) expression in ovarian cancer cells and tumors. In conclusion, the present study showed novel data that CMP could induce ferroptotic death in ovarian cancer cells by suppressing Nrf2/HO-1/xCT/GPX4. All these findings indicate that CMP may have great potential in anti-ovarian cancer cell therapy by inducing ferroptosis.

Prostaglandin E2 Pathway Is Dysregulated in Gastric Adenocarcinoma in a Caucasian Population.

Gastric cancer (GC) represents the third leading cause of cancer-related deaths worldwide. The levels of prostaglandin E2, a key player in the hallmarks of cancer, are mainly regulated by prostaglandin-endoperoxide synthase 2 (PTGS2) and ATP-binding cassette subfamily C member 4 (ABCC4), involved in its synthesis and exportation, respectively, and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and solute carrier organic anion transporter family member 2A1 (SLCO2A1), responsible for its inactivation. Even though there are distinct molecular signatures across ethnic populations, most published studies focus on Asian populations. Our main aim was to explore the genetic expression of the aforementioned molecules in a Caucasian population. 94 “Normal” and 89 tumoral formalin-fixed paraffin-embedded (FFPE) samples from GC patients were used to assess the mRNA expression of PTGS2, ABCC4, hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD), SLCO2A1 by Real-Time PCR. We found an upregulation for the PTGS2 gene mean factor of 2.51 and a downregulation for the HPGD and SLCO2A1 genes (mean factor of 0.10 and 0.37, respectively) in tumorous mucosa in a gender-independent manner. In females, we observed an ABCC4 downregulation and a PTGS2 mRNA upregulation compared to males in tumoral mucosa (mean factor of 0.61 and 1.64, respectively). We reported dysregulation of the inflammation triggered PGE2 pathway in a Caucasian population with an intermediate risk for GC, which might highlight the applicability of aspirin in the treatment of GC patients.

Prognostic Significance of Prostaglandin-Endoperoxide Synthase-2 Expressions in Human Breast Carcinoma: A Multiomic Approach.

Prostaglandin-endoperoxide synthase-2 (PTGS2) plays a pivotal role in inflammation and carcinogenesis in human breast cancer. Our aim of the study is to find the prognostic value of PTGS2 in breast cancer. We conducted a multiomic analysis to determine whether PTGS2 functions as a prognostic biomarker in human breast cancer. We explored PTGS2 mRNA expressions using different public bioinformatics portals. Oncomine, Serial Analysis of Gene Expression (SAGE), GEPIA, ULCAN, PrognoScan database, Kaplan-Meier Plotter, bc-GenExMiner, USC XENA, and Cytoscape/STRING DB were used to identify the prognostic roles of PTGS2 in breast cancer. Based on the clinicopathological analysis, decreased PTGS2 expressions correlated positively with older age, lymph node status, the human epidermal growth factor receptor 2 (HER2) status (P < .0001), estrogen receptor (ER+) expression (P < .0001) Luminal A (P < .0001), and Luminal B (P < .0001). Interestingly, progesterone receptor (PR) (P < .0001) negative showed a high expression of PTGS2. Prostaglandin-endoperoxide synthase-2 was downregulated in breast cancer tissues than in normal tissues. In the PrognoScan database and, Kaplan-Meier Scanner, downregulated expressions of PTGS2 associated with poor overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival. The methylation levels were significantly higher in the Luminal B subtype. Through oncomine coexpressed gene analysis, we found a positive correlation between PTGS2 and interleukin-6 (IL-6) expression in breast cancer tissues. These results indicate that downregulated expressions of PTGS2 can be used as a promising prognostic biomarker and Luminal B hyper methylation may play an important role in the development of breast cancers. However, to clarify our results, extensive study is required.

Labisia pumila prevented osteoarthritis cartilage degeneration by attenuating joint inflammation and collagen breakdown in postmenopausal rat model.

The tropical herb Labisia pumila is traditionally used in facilitating childbirth and post-partum care. The effects of L. pumila leaf extract (LP) in explant cartilage culture and on postmenopausal osteoarthritis (OA) rat model were assessed. The LP (10, 25 and 50µg/ml) or diclofenac (10µg/ml) was added to the cartilage explants containing bovine IL-1β (20ng/ml), to evaluate their direct effects on cartilage degradation. In the preclinical study, rats were grouped (n=8) into: non-treated OA; OA+diclofenac (5mg/kg); OA+LP extract (150 and 300mg/kg); and healthy control. To induce OA, monosodium iodoacetate was injected into the ovariectomised female rats' intra-articular knee joints and evaluated for OA severity after 8weeks via physical (radiological, macroscopic and histological observations), biochemical, ELISA and mRNA expression analysis (for inflammation and cartilage degradation biomarkers). The LP reduced the nitric oxide and proteoglycan release from the cartilage explants under IL-1β induction. The radiological, macroscopic, microscopic and histological images showed the OA rats treated with LP and diclofenac had significantly reduced osteophytes and cartilage erosions compared to non-treated OA rats. The extract significantly up-regulated the anti-inflammatory interleukin-10, collagen type II and down-regulated pro-inflammatory PTGS2 (prostaglandin-endoperoxide synthase 2) mRNA expressions compared to non-treated control. The LP treatment significantly reduced serum collagenases (MMP-1 and MMP-3) and collagen type II degradation biomarker (CTX-II) levels in OA rats. The LP containing myricetin and gallic acid suppressed inflammation, collagenases and cartilage degradation, and helped cartilage matrix synthesis, to prevent OA at the dose equivalent to 30-60mg/kg daily for humans.

Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10–11 (spherical and tubular conceptuses), 11–12 (filamentous conceptuses), 13–14, and 15–16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10–14 of gestation, but was greater on Days 15–16 compared to Days 10–11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11–12 compared to spherical and tubular conceptuses from Days 10–11 (P < 0.01), followed by a decrease on Days 13–14 and 15–16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11–12 than in spherical and tubular conceptuses from Days 10–11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11–12 compared to Days 13–14 and 15–16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11–12 compared to Days 10–11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation.

The expression of genes involved in myometrial contractility changes during ex situ culture of pregnant human uterine smooth muscle tissue.

Background: Ex situ analyses of human myometrial tissue has been used to investigate the regulation of uterine quiescence and transition to a contractile phenotype. Following concerns about the validity of cultured primary cells, we examined whether myometrial tissue undergoes culture-induced changes ex situ that may affect the validity of in vitro models. Objectives: To determine whether human myometrial tissue undergoes culture-induced changes ex situ in Estrogen receptor 1 (ESR1), Prostaglandin-endoperoxide synthase 2 (PTGS2) and Oxytocin receptor (OXTR) expression. Additionally, to determine whether culture conditions approaching the in vivo environment influence the expression of these key genes. Methods: Term non-laboring human myometrial tissues were cultured in the presence of specific treatments, including; serum supplementation, progesterone and estrogen, cAMP, PMA, stretch or NF-κB inhibitors. ESR1, PTGS2 and OXTR mRNA abundance after 48 h culture was determined using quantitative RT-PCR. Results: Myometrial tissue in culture exhibited culture-induced up-regulation of ESR1 and PTGS2 and down-regulation of OXTR mRNA expression. Progesterone prevented culture-induced increase in ESR1 expression. Estrogen further up-regulated PTGS2 expression. Stretch had no direct effect, but blocked the effects of progesterone and estrogen on ESR1 and PTGS2 expression. cAMP had no effect whereas PMA further up-regulated PTGS2 expression and prevented decline of OXTR expression. Conclusion: Human myometrial tissue in culture undergoes culture-induced gene expression changes consistent with transition toward a laboring phenotype. Changes in ESR1, PTGS2 and OXTR expression could not be controlled simultaneously. Until optimal culture conditions are determined, results of in vitro experiments with myometrial tissues should be interpreted with caution.

Endometrial mRNA expression of selected pro-inflammatory factors and mucins in repeat breeder cows with and without subclinical endometritis

Repeat breeder cows (RBC) are defined as cyclic cows without clinical abnormalities that fail to conceive after at least three subsequent inseminations. Previous studies have elucidated cellular defence mechanisms in the bovine uterus but detailed information on inflammatory events of endometrial cells in RBC is still lacking. Thus, the objective of this study was to analyse endometrial mRNA expression of selected transcripts associated with uterine inflammatory processes. Cytobrush samples from 91 RBC and 11 synchronised heifers with no history of gynaecological abnormalities (controls, CON) were collected. The proportion of polymorphonuclear neutrophils in these samples was used for the diagnosis of subclinical endometritis (SE). Ultrasonography and progesterone blood concentrations were used to determine ovarian activity and the stage of the oestrous cycle. Total RNA was isolated from the cytobrush samples and subjected to reverse transcription-quantitative PCR for interleukins (IL) 1A, IL1B, IL6, IL8, chemokine CXL ligand (CXCL) 3, CXCL5, prostaglandin-endoperoxide synthase 2 (PTGS2), tracheal antimicrobial peptide (TAP) and mucin (MUC) 4, MUC5, MUC6, MUC12 and MUC16. CXCL3 mRNA was higher (2-fold) and PTGS2 mRNA lower (6-fold) expressed in RBC compared with CON (P < 0.05). After subdivision of RBC in animals with (RBC-SE) and without SE (RBC-noSE), these differences remained significant between RBC-noSE and CON. Higher mRNA abundances of IL1A and IL1B were found in RBC-SE compared with RBC-noSE (3- and 4-fold; P < 0.05). No differences in the mRNA expression of IL6, IL8, CXCL5 and TAP were observed between RBC-SE, RBC-noSE and CON. MUC4 and MUC12 mRNA was more highly expressed in RBC than in CON (P < 0.05). In RBC-noSE, a 5- and 14-fold higher MUC4 and MUC12 mRNA expression was noticed compared with CON (P < 0.05). A significantly lower mRNA expression of MUC5 and MUC16 (7- and 4-fold) was detected in RBC in the luteal phase compared with RBC in the follicular phase, whereas such a down-regulation was not observed for MUC4 and MUC12. In conclusion, we demonstrated different PTGS2 and CXCL3 mRNA expression between RBC and control heifers, which might be related to subfertility in RBC. Further studies are required to confirm that an unregulated MUC4 and MUC12 mRNA expression may contribute to subfertility of RBC. These findings provide a valid basis for further research on regulatory mechanisms of mRNA expression in subfertile cows.

RAF1 is increased in labouring myometrium and modulates inflammation-induced pro-labour mediators.

Inflammation plays a central role in the terminal process of human labour and delivery, including myometrial contractions. RAF1 proto-oncogene serine/threonine-protein kinase (RAF1) can activate ERK (official gene symbol MAPK1) and/or nuclear factor-kappa B (NF-κB) to regulate genes involved in inflammation. There are, however, no studies on the role of RAF1 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour and pro-inflammatory cytokines interleukin 1 beta (IL1B) and tumour necrosis factor (TNF) alpha on RAF1 protein expression in myometrium and ii) siRNA knockdown of RAF1 on pro-inflammatory and pro-labour mediators in human myometrial primary cells. Term labour was associated with an increase in RAF1 protein expression. Furthermore, RAF1 protein expression was increased in myometrial cells treated with IL1B and TNF, two likely factors contributing to preterm birth. Knockdown of RAF1 by siRNA in primary myometrial cells significantly decreased IL1B- and TNF-induced IL1A, IL1B, IL6, (C-X-C motif) ligand 8 (CXCL8)and chemokine (C-C motif) ligand 2 (CCL2) mRNA abundance and IL6, IL8 and CCL2; prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels and prostaglandin PGF2 α release; and NF-κB activation. Furthermore, RAF1 knockdown was associated with decreased activation of ERK in the presence of IL1B but not TNF. Concordantly, the ERK inhibitor U0126 significantly decreased IL1B-induced IL6, CXCL8, CCL2 and PTGS2 mRNA abundance; IL6, CXCL8, CCL2 and PGF2 α release; and NF-κB activation. In conclusion, IL1B induces the expression and secretion of pro-labour mediators through the RAF1-MAPK1-NF-κB signalling pathway. TNF, on the other hand, regulates pro-labour mediators through the RAF1-NF-κB signalling pathway via an MAPK1-independent mechanism.

Expression of genes involved in the embryo–maternal interaction in the early-pregnant canine uterus

Although there is no acute luteolytic mechanism in the absence of pregnancy in the bitch, a precise and well-timed embryo-maternal interaction seems to be required for the initiation and maintenance of gestation. As only limited information is available about these processes in dogs, in this study, the uterine expression of possible decidualization markers was investigated during the pre-implantation stage (days 10-12) of pregnancy and in the corresponding nonpregnant controls. In addition, the expression of selected genes associated with blastocyst development and/or implantation was investigated in embryos flushed from the uteri of bitches used for this study (unhatched and hatched blastocysts). There was an upregulated expression of prolactin receptor (PRLR) and IGF2 observed pre-implantation. The expression of PRL and of IGF1 was unaffected, and neither was the expression of progesterone- or estrogen receptor β (ESR2). In contrast, (ESR1) levels were elevated during early pregnancy. Prostaglandin (PG)-system revealed upregulated expression of PGE2-synthase and its receptors, PTGER2 and PTGER4, and of the PG-transporter. Elevated levels of AKR1C3 mRNA, but not the protein itself, were noted. Expression of prostaglandin-endoperoxide synthase 2 (PTGS2) remained unaffected. Most of the transcripts were predominantly localized to the uterine epithelial cells, myometrium and, to a lesser extent, to the uterine stroma. PGES (PTGES) mRNA was abundantly expressed in both groups of embryos and appeared higher in the hatched ones. The expression level of IGF2 mRNA appeared higher than that of IGF1 mRNA in hatched embryos. In unhatched embryos IGF1, IGF2, and PTGS2 mRNA levels were below the detection limit.

Absence of seasonal changes in FSHR gene expression in the cat cumulus–oocyte complex in vivo and in vitro

Domestic cat oocytes are seasonally sensitive to FSH. Compared with those collected during the breeding season, oocytes from the nonbreeding (NB) season require more FSH during in vitro maturation to achieve comparable developmental competence. This study tested the hypothesis that this seasonal variation was due to altered expression of FSH receptors (FSHR) and/or FSH-induced genes. Relative expression levels of FSHR mRNA and FSH-enhanced gene estrogen receptor β (ESR2) were measured by qPCR in whole ovaries and immature cumulus-oocyte complexes (COCs) isolated from cat ovaries during the natural breeding vs NB seasons. Expression levels of FSH-induced genes prostaglandin-endoperoxide synthase 2 (PTGS2), early growth response protein-1 (EGR1), and epidermal growth factor receptor (EGFR) were examined in mature COCs from both seasons that were a) recovered in vivo or b) matured in vitro with conventional (1 μg/ml) or high (10 μg/ml) FSH concentrations. Overall, FSHR mRNA levels were lower in whole ovaries during the NB compared with breeding season but were similar in immature COCs, whereas ESR2 levels did not differ in either group between intervals. We observed changes in PTGS2, EGR1, and EGFR mRNA expression patterns across maturation in COCs within but not between the two seasons. The lack of seasonal differentiation in FSH-related genes was not consistent with the decreased developmental capacity of oocytes fertilized during the NB season. These findings reveal that the seasonal decrease in cat oocyte sensitivity to FSH occurs both in vivo and in vitro. Furthermore, this decline is unrelated to changes in expression of FSHR mRNA or mRNA of FSH-induced genes in COCs from antral follicles.

Role of Angiotensin II in the Periovulatory Epidermal Growth Factor-Like Cascade in Bovine Granulosa Cells In Vitro1

Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.

PTGS2 (Prostaglandin Endoperoxide Synthase-2) Expression in Term Human Amnion in Vivo Involves Rapid mRNA Turnover, Polymerase-II 5′-Pausing, and Glucocorticoid Transrepression

The in vivo role of glucocorticoids in controlling prostaglandin endoperoxide synthase-2 (PTGS2) expression in the human amnion is unclear despite extensive studies using in vitro models. We addressed this issue by determining PTGS2 mRNA levels and gene transcriptional activity, RNA polymerase-II (pol-II) binding, pol-II C-terminal domain (CTD) phosphorylation, histone acetylation, and histone methylation at the PTGS2 gene in fresh amnion and in amnion explants incubated with dexamethasone for 24 h after delivery, when adaptation from in vivo to in vitro conditions occurred. PTGS2 mRNA turnover changed during incubation involving the initial rapid decrease and subsequent rebound of the transcription rate and stabilization of mRNA. pol-II accumulated in the 5'-region of the gene, which indicated postinitiation pausing. pol-II binding, 5'-accumulation, C-terminal domain Ser-5 and Ser-2 phosphorylation, and histone acetylation decreased rapidly and did not reverse during the transcriptional rebound, suggesting that the transcriptional mechanism altered in vitro. Dexamethasone decreased PTGS2 gene activity and mRNA levels. Glucocorticoid receptor-α (GRα) was bound to the PTGS2 promoter but did not affect pol-II recruitment, pausing, or the epigenetic marks. GRα binding, however, decreased initiating (Ser-5) and elongating (Ser-2) pol-II phosphorylation. The ability of the PTGS2 promoter to bind GRα in response to dexamethasone diminished during incubation. We conclude that PTGS2 mRNA turnover is accelerated in vivo, but the underlying mechanisms are not sustained beyond 24 h in explants. Glucocorticoids chronically transrepress PTGS2 gene activity in vivo in part by interfering with transcription initiation and elongation. Glucocorticoid transrepression of PTGS2 may be important for pregnancy maintenance and the timing of parturition.

Evidence for a Spatial and Temporal Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion in Term and Preterm Parturition

Prostaglandin-endoperoxide synthase 2 (PTGS2) is a key enzyme involved in parturition. PTGS2 mRNA was found to be differentially expressed between placental amnion (amnion overlying the placental disc) and reflected amnion (amnion of the extraplacental chorioamniotic membranes) in term placentas. The aim was to evaluate the spatial and temporal regulation of PTGS2 expression in the amnion and the chorion-decidua. PTGS2 expression was analyzed in the amnion and chorion-decidua obtained from 32 women: term not in labor (n = 12), term in labor (n = 12), and preterm labor (n = 8), by immunoblotting and densitometry. Prostaglandin E(2) (PGE(2)) in the amnion and chorion-decidua was measured by a specific immunoassay. Compared to preterm labor cases, PTGS2 expression increased at term before the onset of labor far more prominently in placental amnion (4.5-fold; P = 0.002) than in reflected amnion (1.4-fold; P = 0.007). There was a significant increase in PTGS2 expression in reflected amnion (2.9-fold; P < 0.01) but not in placental amnion with labor at term. PTGS2 expression was higher in reflected amnion than in chorion-decidua in labor at term (2.9-fold; P < 0.01). PTGS2 was barely detected in amnion and chorion-decidua with preterm labor. Expression of PGE(2) showed a good correlation with PTGS2 expression (r = 0.722; P < 0.001). PTGS2 expression in the amnion shows a distinct spatial and temporal regulation. Spontaneous labor at term and pathological preterm labor clearly differ in amniotic PTGS2 and PGE(2) abundance. Our observations underscore the biological significance of the amnion and amniotic fluid in human parturition.

Differential Expression of the Enzymatic System Controlling Synthesis, Metabolism, and Transport of PGF2 Alpha in Human Fetal Membranes1

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum

BackgroundInflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1β (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period.MethodsEndometrial samples were obtained from primiparous cows (n = 5) on days 10, 17, 24, 31, 38 and 45 postpartum (pp) using the cytobrush technique. Cytological smears were prepared from cytobrush samples to determine the proportion of polymorphonuclear neutrophils (PMN). Total RNA was extracted from endometrial samples, and real-time RT-PCR was performed.ResultsA time-dependent mRNA expression of the investigated factors was found for the course of the postpartum period. In detail, a significantly higher expression of these factors was observed on day 17 pp compared to day 31 pp. Furthermore, the proportion of PMN peaked between days 10-24 pp and decreased thereafter to low percentages (< 5%) on day 31 pp and thereafter. In addition, CXCL5, IL1B, IL8 and HP mRNA expression correlated significantly with the proportion of PMN (P < 0.05). A significantly higher CXCL5, IL1B, IL6, IL8, PTGS2 and TNF mRNA content was observed in samples from cows with an inflamed endometrium compared with samples from cows with a healthy endometrium (P < 0.05).ConclusionsThese results show that inflammatory cytokines and acute phase proteins are expressed in the bovine endometrium in a time-related manner during the postpartum period, with a significant expression peak on day 17 pp as a possible mucosal immune response in the uterus. The evaluation of the expression patterns of such candidate genes may reveal more information than only determining the percentage of PMN to judge the severity of an inflammation.

Analysis of Lysophosphatidic Acid (LPA) Receptor and LPA-Induced Endometrial Prostaglandin-Endoperoxide Synthase 2 Expression in the Porcine Uterus

Lysophosphatidic acid (LPA), a simple phospholipid-derived mediator with diverse biological actions, acts through the specific G protein-coupled receptors endothelial differentiation gene (EDG) 2, EDG4, EDG7, and GPR23. Recent studies indicate a critical role for LPA receptor signaling in embryo implantation. To understand how LPA acts in the uterus during pregnancy in pigs, we evaluated: 1) spatial and temporal expression of LPA receptors in the uterine endometrium during the estrous cycle and pregnancy and in early-stage concepti, 2) LPA levels in uterine luminal fluids from d 12 of the estrous cycle and pregnancy, 3) effects of steroid hormones on EDG7 mRNA levels, and 4) effects of LPA on prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in the uterine endometrium using explant cultures. Of the four receptors, EDG7 was dominant, and its expression was regulated by pregnancy stage and status. EDG7 expression was highest on d 12 pregnancy, and localized to the luminal and glandular epithelium, and EDG7 mRNA levels were elevated by estrogen in the endometrium. EDG7 expression was also detected in concepti of d 12 and 15. LPA with various fatty acyl groups was present in the uterine lumen on d 12 of both the estrous cycle and pregnancy. LPA increased PTGS2 mRNA abundance in the uterine endometrium. These results indicate that LPA produced in the uterine endometrium may play a critical role in uterine endometrial function and conceptus development through EDG7-mediated PTGS2 expression during implantation and establishment of pregnancy in pigs.

Opposite Effect of Phorbol Ester PMA on PTGS2 and PGDH mRNA Expression in Human Chorion Trophoblast Cells

Prostaglandins (PGs) induce the mechanism of labor in humans. The enzymes responsible for PG synthesis and metabolism are prostaglandin-endoperoxide synthase 2 (PTGS2) and 15-hydroxyprostaglandin dehydrogenase (PGDH). In human chorion trophoblast cells, calcium ionophore A23187 upregulates PTGS2 and downregulates PGDH protein and mRNA. The authors hypothesize that this regulation requires activation of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs). Human chorion trophoblasts were incubated with A23187 or phorbol 12-myristate 13-acetate (PMA) in the absence or presence of inhibitors of PKC, c-Jun N-terminal kinase, p38, and MEK1/2. PTGS2 and PGDH mRNA were measured by real-time reverse-transcription polymerase chain reaction. PMA upregulated PTGS2 and downregulated PGDH. The PMA effect was reversed by the inhibition of PKC. The p38 inhibitor reduced the stimulatory effect of PMA and A23187 on PTGS2. MEK1/2 inhibitor reduced the effect of PMA on PTGS2. All MAPK inhibitors failed to reverse the effect of either A23187 or PMA on PGDH. The authors conclude that upon stimulation with the same upstream signals, different downstream intracellular pathways regulate PTGS2 and PGDH mRNA expression.

The Expression of Prostaglandin Endoperoxide Synthase 2 Messenger RNA and the Proportion of Smooth Muscle and Collagen in the Sheep Cervix During the Estrous Cycle

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

Expression of Prostaglandin-Endoperoxide Synthase 1 and Prostaglandin-Endoperoxide Synthase 2 in Human Osteoblasts

A Prostaglandin endoperoxide synthase isoenzyme was recently identified in several cell lines. Osteoblasts possess Prostaglandin endoperoxide synthase activity, but it is not known which isoenzymes are present in these cells. Our objective was to identify these isoenzymes in human osteoblasts. Resting cells in culture did not produce measurable amounts of PGE 2 and did not express Prostaglandin endoperoxide synthase-1 or Prostaglandin endoperoxide synthase-2 mRNAs detectable by Northern blot. Treatment with rhIL1α or rhTNFα induced both the expression of Prostaglandin endoperoxide synthase-2 mRNA and the synthesis of PGE 2, rhIL-1α being more potent on an equimolar basis than rhTNFα. Dexamethasone inhibited the increase in Prostaglandin endoperoxide synthase-2 mRNA and the production of PGE 2 induced by both cytokines. These results suggest that Prostaglandin endoperoxide synthase-2 may be the relevant isoenzyme for prostanoid production in human osteoblasts in culture.