Expression data from primary culture human myometrial cells

Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone's anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays (GSE68171) to find P4 and IL-1β responsive genes and IL-1β responsive genes which were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1β in human myometrial cells. These data will also help us understand the role of PR and GR in human parturition

Chemical profiles and simultaneous quantification of Aurantii fructus by use of HPLC-Q-TOF-MS combined with GC-MS and HPLC methods

Aurantii fructus (AF) is a traditional Chinese medicine that has been used to improve gastrointestinal motility disorders for over a thousand years, but there is no exhaustive identification of the basic chemical components and comprehensive quality control of this herb. In this study, high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) and gas chromatography coupled mass spectrometry (GC-MS) were employed to identify the basic chemical compounds, and high-performance liquid chromatography (HPLC) was developed to determine the major biochemical markers from AF extract. There were 104 compounds belonging to eight structure types, including 13 amino acids or peptides, seven alkaloids, 18 flavanones, 14 flavones, 15 polymethoxyflavonoids, six triterpenoids, nine coumarins, and 18 volatile oils, as well as four other compounds that were systematically identified as the basic components from AF, and among them, 41 compounds were reported for the first time. Twelve bioactive ingredients were chosen as the benchmark markers to evaluate the quality of AF. The analysis was completed with a gradient elution at a flow rate of 0.7 mL/min within 55 min. This efficient method was validated showing good linearity, precision, stability, repeatability and recovery. Furthermore, the method was successfully applied to the simultaneous determination of 12 chemical markers in different samples of AF. This study could be applied to the identification of multiple bioactive substances and improve the quality control of AF

PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour

Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1β-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1β-induced COX-2 mRNA expression and reduced the IL-1β-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1β increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1β-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1β, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility

Placental syncytiotrophoblast constitutes a major barrier to vertical transmission of Listeria monocytogenes.

Listeria monocytogenes is an important cause of maternal-fetal infections and serves as a model organism to study these important but poorly understood events. L. monocytogenes can infect non-phagocytic cells by two means: direct invasion and cell-to-cell spread. The relative contribution of each method to placental infection is controversial, as is the anatomical site of invasion. Here, we report for the first time the use of first trimester placental organ cultures to quantitatively analyze L. monocytogenes infection of the human placenta. Contrary to previous reports, we found that the syncytiotrophoblast, which constitutes most of the placental surface and is bathed in maternal blood, was highly resistant to L. monocytogenes infection by either internalin-mediated invasion or cell-to-cell spread. Instead, extravillous cytotrophoblasts-which anchor the placenta in the decidua (uterine lining) and abundantly express E-cadherin-served as the primary portal of entry for L. monocytogenes from both extracellular and intracellular compartments. Subsequent bacterial dissemination to the villous stroma, where fetal capillaries are found, was hampered by further cellular and histological barriers. Our study suggests the placenta has evolved multiple mechanisms to resist pathogen infection, especially from maternal blood. These findings provide a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta

Plasma membrane receptor mediated MAPK signaling pathways are activated in human uterine cervix at parturition

BACKGROUND: Cervical ripening resembles an inflammatory reaction. Estrogens induce leukocyte migration into tissue and factors promoting cervical remodeling and labor, although the mechanisms are only partially known. The aim of this study was to investigate whether plasma membrane receptor mediated pathways, known to be activated by estrogens and proinflammatory compounds, are involved in cervical ripening before labor. METHODS: The expression and distribution of mitogen activated protein kinases (MAPK), which transduce extracellular signals into intracellular responses through phosphorylation, and their intracellular targets transcription factors c-Jun and c-Fos proteins (AP-1) were analysed in cervical biopsies from term pregnant women (TP), immediately after parturition (PP), and from non-pregnant women (NP). Immunohistochemistry and RT-PCR techniques were used. RESULTS: Cell-specific alterations in the immunostaining pattern for MAPK were observed. The expressions of activated, phosphorylated MAPK forms pERK1/2, pJNK and p38MAPK were significantly increased in cervical stroma until TP and pERK1/2 expression was significantly enhanced in PP group. c-Jun was significantly increased in cervical stroma and smooth muscle in TP as compared to NP group. c-Fos was significantly increased in stroma, squamous epithelium and glandular epithelium in PP as compared to TP group. CONCLUSION: We report, for the first time, cell-specific activation of pMAPKs and their targets transcription factors c-Fos and c-Jun (AP-1) proteins in human uterine cervix until term pregnancy, and immediately after parturition. These results suggest a role for MAPK activation in cervical ripening before labor

Global report on preterm birth and stillbirth (2 of 7): discovery science

<p>Abstract</p> <p>Background</p> <p>Normal and abnormal processes of pregnancy and childbirth are poorly understood. This second article in a global report explains what is known about the etiologies of preterm births and stillbirths and identifies critical gaps in knowledge. Two important concepts emerge: the continuum of pregnancy, beginning at implantation and ending with uterine involution following birth; and the multifactorial etiologies of preterm birth and stillbirth. Improved tools and data will enable discovery scientists to identify causal pathways and cost-effective interventions.</p> <p>Pregnancy and parturition continuum</p> <p>The biological process of pregnancy and childbirth begins with implantation and, after birth, ends with the return of the uterus to its previous state. The majority of pregnancy is characterized by rapid uterine and fetal growth without contractions. Yet most research has addressed only uterine stimulation (labor) that accounts for <0.5% of pregnancy.</p> <p>Etiologies</p> <p>The etiologies of preterm birth and stillbirth differ by gestational age, genetics, and environmental factors. Approximately 30% of all preterm births are indicated for either maternal or fetal complications, such as maternal illness or fetal growth restriction. Commonly recognized pathways leading to preterm birth occur most often during the gestational ages indicated: (1) inflammation caused by infection (22-32 weeks); (2) decidual hemorrhage caused by uteroplacental thrombosis (early or late preterm birth); (3) stress (32-36 weeks); and (4) uterine overdistention, often caused by multiple fetuses (32-36 weeks). Other contributors include cervical insufficiency, smoking, and systemic infections. Many stillbirths have similar causes and mechanisms. About two-thirds of late fetal deaths occur during the antepartum period; the other third occur during childbirth. Intrapartum asphyxia is a leading cause of stillbirths in low- and middle-income countries.</p> <p>Recommendations</p> <p>Utilizing new systems biology tools, opportunities now exist for researchers to investigate various pathways important to normal and abnormal pregnancies. Improved access to quality data and biological specimens are critical to advancing discovery science. Phenotypes, standardized definitions, and uniform criteria for assessing preterm birth and stillbirth outcomes are other immediate research needs.</p> <p>Conclusion</p> <p>Preterm birth and stillbirth have multifactorial etiologies. More resources must be directed toward accelerating our understanding of these complex processes, and identifying upstream and cost-effective solutions that will improve these pregnancy outcomes.</p

Cyclic AMP effectors regulate myometrial oxytocin receptor expression

The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation

Transforming growth factor beta 1 induced endothelin-1 release is peroxisome proliferator-activated receptor gamma dependent in A549 cells.

Endothelin-1 (ET-1) is involved in pulmonary vascular remodeling. The aim of this study was to investigate the biochemical interactions between PPAR-γ, TGF-β1 and ET-1 in vitro.A549 cells were pre-treated with S2505 (10 μM), S2871 (10 μM) with/without SB203580 (10 μM) for 60 min following 2 h treatment with 10 ng/mL TGF-β1. A549 cells were also transfected with positive or negative PPAR-γ plasmids for comparison. RT-PCR, ELISA, western blotting and confocal laser scanning microscopy (CLSM) were used to measure the relevant expression of mRNA, protein, mediators of pathways and nuclear factor translocation.SB203580 inhibited TGF-β1 induced ET-1 expression in A549 cells. S2871 decreased PPAR-γ mRNA and increase TGF-β1-induced ET-1 expression. S2871 increased phosphorylation of p38 MAPK and Smad2. Cells transfected with PPAR-γ negative plasmid increased TGF-β1 induced ET-1 expression, and increased the expression of phospho-p38 MAPK and phospho-Smad2. S2505 increased PPAR-γ mRNA expression, suppressed the increased TGF-β1-induced expression of ET-1. S2505 inhibited TGF-β1 induced phosphorylation of p38 MAPK and Smad2, also the nuclear translocation of Smad2. Cells transfected with PPAR-γ positive plasmid reduced TGF-β1-induced ET-1 expression, and inhibited the expression of phospho-p38 MAPK and phospho-Smad2.TGF-β1 induced release of endothelin-1 is PPAR-γ dependent in cultured A549 cells

Is there an inflammatory stimulus to human term labour?

Inflammation is thought to play a pivotal role in the onset of term and some forms of preterm labour. Although, we recently found that myometrial inflammation is a consequence rather than a cause of term labour, there are several other reproductive tissues, including amnion, choriodecidua parietalis and decidua basalis, where the inflammatory stimulus to labour may occur. To investigate this, we have obtained amnion, choriodecidual parietalis and decidua basalis samples from women at various stages of pregnancy and spontaneous labour. The inflammatory cytokine profile in each tissue was determine by Bio-Plex Pro® cytokine multiplex assays and quantitative RT-PCR. Active motif assay was used to study transcription activation in the choriodecidua parietalis. Quantitative RT-PCR was use to study the pro-labour genes (PGHS-2, PGDH, OTR and CX43) in all of the tissues at the onset of labour and oxytocin (OT) mRNA expression in the choriodecidual parietalis and decidua basalis. Statistical significance was ascribed to a P value <0.05. In the amnion and choriodecidua parietalis, the mRNA levels of various cytokines decreased from preterm no labour to term no labour samples, but the protein levels were unchanged. The choriodecidua parietalis showed increase in the protein levels of IL-1β and IL-6 in the term early labour samples. In the amnion and decidua basalis, the protein levels of several cytokines rose in term established labour. The multiples of the median derived from the 19-plex cytokine assay were greater in term early labour and term established labour samples from the choriodecidua parietalis, but only in term established labour for myometrium. These data suggest that the inflammatory stimulus to labour may begin in the choriodecidua parietalis, but the absence of any change in prolabour factor mRNA levels suggests that the cytokines may act on the myometrium where we observed changes in transcription factor activation and increases in prolabour gene expression in earlier studies