The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

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Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour

PLoS ONE ◽

10.1371/journal.pone.0240325 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0240325

Author(s):

Angela Yulia ◽

Alice J. Varley ◽

Natasha Singh ◽

Kaiyu Lei ◽

Rachel Tribe ◽

...

Keyword(s):

Preterm Labour ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Oxytocin Receptor ◽

Receptor Expression ◽

Mrna Levels ◽

Effector Pathway ◽

Effector System ◽

Term Labour ◽

Camp Levels

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined. Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour. These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

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Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro

Biochemical Journal ◽

10.1042/bj3560883 ◽

2001 ◽

Vol 356 (3) ◽

pp. 883-889 ◽

Cited By ~ 68

Author(s):

Lorraine GAMBLING ◽

Ruth DANZEISEN ◽

Susan GAIR ◽

Richard G. LEA ◽

Zehane CHARANIA ◽

...

Keyword(s):

Iron Deficiency ◽

Transferrin Receptor ◽

Iron Transport ◽

Receptor Expression ◽

Mrna Levels ◽

Iron Transfer ◽

Metal Transporter ◽

Protein Levels ◽

Copper Oxidase ◽

Maternal Iron

Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine (‘deferioxamine’), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.

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Regulation of the Endothelin/Endothelin Receptor System by Interleukin-1β in Human Myometrial Cells

Endocrinology ◽

10.1210/en.2005-0250 ◽

2005 ◽

Vol 146 (11) ◽

pp. 4878-4886 ◽

Cited By ~ 9

Author(s):

Michelle Breuiller-Fouché ◽

Catherine Morinière ◽

Emmanuelle Dallot ◽

Stéphanie Oger ◽

Régis Rebourcet ◽

...

Keyword(s):

Prolonged Exposure ◽

Receptor Expression ◽

Receptor Subtype ◽

Mrna Levels ◽

Receptor System ◽

Myometrial Cell ◽

Myometrial Cells ◽

Uterine Contractions ◽

Eta Receptor ◽

Etb Receptor

Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1β, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1β on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1β led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1β treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1β abolished the contractile effect induced by ET-1. Such a regulation of IL-1β on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.

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HIV-1 Tat Regulates Occludin and AβTransfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/4196572 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Yanlan Chen ◽

Wen Huang ◽

Wenlin Jiang ◽

Xianghong Wu ◽

Biao Ye ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Receptor Expression ◽

Immunofluorescent Staining ◽

Mrna Levels ◽

Protein Levels ◽

Density Lipoprotein Receptor ◽

Hiv 1

HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβtransfer receptors.

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Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00577.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. E857-E863 ◽

Cited By ~ 12

Author(s):

Anastasia Kariagina ◽

Svetlana Zonis ◽

Mahta Afkhami ◽

Dmitry Romanenko ◽

Vera Chesnokova

Keyword(s):

Glucocorticoid Receptor ◽

Hpa Axis ◽

Leukemia Inhibitory Factor ◽

Dominant Negative ◽

Receptor Expression ◽

Inhibitory Factor ◽

Mrna Levels ◽

Hypothalamic Pituitary Adrenal ◽

Protein Levels

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 μg/mouse, ip) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

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Transcriptional Inhibition of Oxytocin Receptor Expression in Human Myometrial Cells by Melatonin Involves Protein Kinase C Signaling

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2007-1128 ◽

2007 ◽

Vol 92 (10) ◽

pp. 4015-4019 ◽

Cited By ~ 17

Author(s):

James Sharkey ◽

James Olcese

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Gene Transcription ◽

Late Pregnancy ◽

Oxytocin Receptor ◽

Receptor Expression ◽

Human Myometrium ◽

Myometrial Cells ◽

Kinase C

Abstract Context: Our laboratory recently characterized the expression of the melatonin receptors in the human myometrium and showed that the expression of these receptors is suppressed during late pregnancy. Objective: In an effort to understand better the significance of melatonin in the human myometrium, we explored the mechanisms through which this hormone influences the expression of the oxytocin receptor in vitro. Design: The stable melatonin analog iodomelatonin was presented to cultured telomerase-immortalized myometrial cells of the human telomerase reverse transcriptase line under physiological doses and durations. Pharmacological inhibitors of melatonin binding, gene transcription, phospholipase C, and protein kinase C signaling were used to define the mechanism of melatonin action. Results: Our results reveal that melatonin significantly inhibits oxytocin receptor mRNA expression primarily via the melatonin 2 receptor. The melatonin-dependent decrease in oxytocin receptor transcripts involves suppression of gene transcription rather than enhanced rates of transcript degradation. Melatonin effects were abolished by pretreating the cells with the phospholipase C inhibitor U73122 or the protein kinase C inhibitor C1. Conclusions: Melatonin, like oxytocin, can negatively regulate oxytocin receptor transcription in human myometrial cells via modulation of protein kinase C signaling. This is consistent with the hypothesis that the reduced melatonin receptor expression during late pregnancy, which occurs at a time when oxytocin receptors are up-regulated, may be physiologically important for the subsequent timing of labor.

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Ontogeny and effect of cortisol on vasopressin-1b receptor expression in anterior pituitaries of fetal sheep

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00427.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. R51-R56 ◽

Cited By ~ 7

Author(s):

Sharla F. Young ◽

Jennifer L. Smith ◽

Jorge P. Figueroa ◽

James C. Rose

Keyword(s):

Receptor Expression ◽

Late Gestation ◽

Mrna Levels ◽

Fetal Sheep ◽

Fetal Plasma ◽

Protein Levels ◽

Receptor Mrna ◽

Naturally Occurring ◽

V1b Receptor

Corticotroph responsiveness to arginine vasopressin (AVP) increases during late gestation in fetal sheep. The mechanism of this increase in AVP responsiveness is currently unknown but could be related to an increase in vasopressin type 1b (V1b) receptor expression in the pituitary during development. To determine if there are ontogenic changes in V1b receptor expression that may help explain the changes in ACTH responses to AVP, we studied pituitaries from three groups of fetal sheep [100, 120, or 140 days gestational age (dGA)]. V1b receptor mRNA and protein significantly decreased by 140 dGA. Peak V1b mRNA levels were detected at 100 dGA, while peak V1b protein levels were detected at 120 dGA. The reduction in V1b receptor expression in late gestation may be due to the naturally occurring peripartum increase in fetal plasma cortisol because cortisol infusion at 122–130 dGA decreased V1b receptor mRNA. Thus there is a marked decrease in the expression of the V1b receptor in the pituitary during fetal development, leaving the role of the V1b receptor in increasing AVP responsiveness uncertain.

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Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00025.2005 ◽

2005 ◽

Vol 289 (2) ◽

pp. R410-R417 ◽

Cited By ~ 9

Author(s):

Nancy K. Valego ◽

Yixin Su ◽

Luke C. Carey ◽

Sharla F. Young ◽

Stephen B. Tatter ◽

...

Keyword(s):

Plasma Cortisol ◽

Receptor Expression ◽

Late Gestation ◽

Mrna Levels ◽

Acth Stimulation ◽

Fetal Sheep ◽

Protein Levels ◽

Overnight Incubation ◽

Acth Receptor ◽

Underlying Mechanisms

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At ∼138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 ± 1.2 vs. 47.3 ± 11.1 ng·ml−1·2 h−1) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.

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TBX2, a Novel Regulator of Labour

Medicina ◽

10.3390/medicina57060515 ◽

2021 ◽

Vol 57 (6) ◽

pp. 515

Author(s):

Febilla Fernando ◽

Geertruda J.M. Veenboer ◽

Martijn A. Oudijk ◽

Marlies A.M. Kampman ◽

Karst Y. Heida ◽

...

Keyword(s):

Preterm Labour ◽

Therapeutic Interventions ◽

Human Myometrium ◽

Mrna Levels ◽

Myometrial Cells ◽

Cytokines And Chemokines ◽

Term Labour ◽

Upstream Regulators

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

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An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

10.1101/588798 ◽

2019 ◽

Author(s):

Ewan K.S. McRae ◽

Steven J. Dupas ◽

Evan P. Booy ◽

Ramanaguru S. Piragasam ◽

Richard P. Fahlman ◽

...

Keyword(s):

Mrna Levels ◽

Wild Type ◽

Biologically Relevant ◽

Protein Levels ◽

Guanine Quadruplex ◽

Repressive Effect ◽

G Quadruplex ◽

Different Levels ◽

In Cells

AbstractDDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we identified 26 proteins that are expressed at significantly different levels in cells expressing wild type DDX21 relative to an rG4 binding deficient DDX21 (M4). From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5’UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing only DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in cells expressing only DDX21 M4, and rendering previously resistant cells sensitive to TRAIL mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by a rG4 forming potential in their 5’UTRs.

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