- Email: [email protected]
pro-labor stimuli
Accepted Manuscript Mechanism by which progesterone and cAMP synergize to maintain uterine quiescence during pregnancy Peyvand Amini, Rachel Wilson, William Koeblitz, Junye Wang, Huiqing Tan, Lijuan Yi, Zachary Stanfield, Andrea Romani, Charles Malemud, Sam Mesiano PII:
S0303-7207(18)30240-5
DOI:
10.1016/j.mce.2018.08.005
Reference:
MCE 10283
To appear in:
Molecular and Cellular Endocrinology
Received Date: 12 June 2018 Revised Date:
13 August 2018
Accepted Date: 13 August 2018
Please cite this article as: Amini, P., Wilson, R., Koeblitz, W., Wang, J., Tan, H., Yi, L., Stanfield, Z., Romani, A., Malemud, C., Mesiano, S., Mechanism by which progesterone and cAMP synergize to maintain uterine quiescence during pregnancy, Molecular and Cellular Endocrinology (2018), doi: 10.1016/j.mce.2018.08.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Mechanism by which Progesterone and cAMP Synergize to Maintain Uterine Quiescence during Pregnancy
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Peyvand Amini3, Rachel Wilson1, William Koeblitz1, Junye Wang1, Huiqing Tan1, Lijuan Yi1, Zachary Stanfield5, Andrea Romani3, Charles Malemud4, Sam Mesiano1,2 1
Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH
2
Department of Obstetrics and Gynecology, University Hospitals Cleveland Medical Center,
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Cleveland OH 3
Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH
4
Department of Medicine, Case Western Reserve University, Cleveland, OH
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5
Systems Biology and Bioinformatics, Case Western Reserve University, Cleveland, OH
Abbreviated title: Synergism between progesterone and cAMP
Key terms: progesterone receptor, cyclic AMP, pregnancy, inflammation, myometrium
Sam Mesiano, PhD
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Corresponding author:
Department of Reproductive Biology
11100 Euclid Ave, Cleveland, OH 44106
Fax: 216-844-7095
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Phone: 216-844-1553
Email: [email protected]
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Grants supporting this study: The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (HD069819). March of Dimes Prematurity Center, Ohio Collaborative.
Disclosure statement: The authors have nothing to disclose
Short title: Anti-inflammatory actions of progesterone and cAMP Page 1
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ABSTRACT Progesterone (P4) acting through the P4 receptor (PR) isoforms, PR-A and PR-B, promotes uterine quiescence for most of pregnancy, in part, by inhibiting the response of myometrial cells
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to pro-labor inflammatory stimuli. This anti-inflammatory effect is inhibited by phosphorylation of PR-A at serine-344 and -345 (pSer344/345-PRA). Activation of the cyclic adenosine monophosphate (cAMP) signaling pathway also promotes uterine quiescence and myometrial relaxation. This study examined the cross-talk between P4/PR and cAMP signaling to exert anti-
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inflammatory actions and control pSer344/345-PRA generation in myometrial cells. In the hTERT-
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HMA/B immortalized human myometrial cell line P4 inhibited responsiveness to interleukin (IL)1ß and this anti-inflammatory effect was increased by forskolin (increases cAMP) and 8-BrcAMP in a concentration-dependent and synergistic manner that was mediated by activation of protein kinase A (PKA). Forskolin also inhibited the generation of pSer344/345-PRA and expression of key contraction-associated genes in a concentration-dependent manner.
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Generation of pSer344/345-PRA was catalyzed by stress-activated protein kinase/c-Jun NH2terminal kinase (SAPK/JNK). Forskolin inhibited pSer344/345-PRA generation, in part, by increasing the expression of dual specificity protein phosphatase 1 (DUSP1), a phosphatase
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that inactivates mitogen-activated protein kinases (MAPKs) including SAPK/JNK. P4/PR and forskolin increased DUSP1 expression. The data suggest that P4/PR promotes uterine
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quiescence via cross-talk and synergy with cAMP/PKA signaling in myometrial cells via DUSP1mediated inhibition of SAPK/JNK activation.
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INTRODUCTION Parturition (the process of birth) involves transition of the uterus from the quiescent to the laboring state wherein the myometrium (the smooth muscle of the uterine wall) produces phasic
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and forceful contractions to become the engine for birth. In women, a major trigger for parturition is tissue-level inflammation in the myometrium that involves the infiltration and activation of immune cells and the local production of pro-inflammatory cytokines, especially interleukin (IL)-
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1ß (IL-1ß) and prostaglandins (PGs) (1-5). For most of pregnancy the steroid hormone progesterone (P4), via its interaction with the nuclear P4 receptor (PR) isoforms, PR-A and PR-
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B, in myometrial cells blocks labor, in part, by inhibiting responsiveness to pro-labor/proinflammatory stimuli (i.e., an anti-inflammatory effect), thus preventing tissue-level inflammation (6-10). In all viviparous species, parturition is induced by the withdrawal of P4/PR signaling and in women this is thought to be mediated by the functional loss of P4/PR anti-inflammatory activity via changes in PR transcriptional activity (11,12). Our previous studies suggest that
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phosphorylation of PR-A at serine residues -344 and -345 (pSer344/345-PRA; numbers relative to amino acid 1 in PR-B) in myometrial cells causes functional P4/PR withdrawal (9). The level of pSer344/345-PRA in term myometrium was markedly higher in laboring compared with non-
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laboring tissue, and that generation of pSer344/345-PRA in myometrial cells was P4-dependent and induced by IL-1ß. Importantly, we also found that the capacity for P4/PR to exert anti-
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inflammatory activity in myometrial cells was inhibited by pSer344/345-PRA. The data suggest that human parturition involves pSer344/345-PRA-mediated functional P4/PR withdrawal and that prevention of pSer344/345-PRA generation in myometrial cells is important to maintain uterine quiescence for most of pregnancy. The 3',5'-cyclic adenosine monophosphate (cAMP) intracellular signaling cascade is a major inhibitor of smooth muscle contractility and is thought to play a key role in maintaining myometrial quiescence during pregnancy (13,14). cAMP is produced by the adenylyl cyclase enzyme which is activated by hormones that bind Gαs-coupled transmembrane receptors. In Page 3
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most cells cAMP functions as a second messenger that activates protein kinase-A (PKA), a multifunctional kinase that phosphorylates downstream targets to induce a variety of cellular response (15). In smooth muscle cells PKA suppresses contractility by inhibiting myosin light
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chain kinase activity and decreasing the intracellular concentration of free Ca2+ (16-21). cAMP may also contribute to the maintenance of pregnancy by exerting anti-inflammatory activity in myometrial cells, and by boosting P4/PR transcriptional activity (8). Studies in airway smooth
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muscle cells show that cAMP signaling inhibits response to pro-inflammatory stimuli by suppressing the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-
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κB) and mitogen-activate protein kinases (MAPKs) (22). In breast cancer cell lines, cAMP affects P4/PR signaling in a quantitative and qualitative manner (23,24), and in myometrial cells derived from term uterus, forskolin, a bicyclic diterpene that increase intracellular cAMP by stimulating adenylyl cyclase, increases the anti-inflammatory activity of P4 (8). Thus, the functional interaction between P4/PR and cAMP signaling in myometrial cells may be critical for
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the maintenance of uterine quiescence during pregnancy by promoting myometrial cell relaxation and refractoriness to pro-inflammatory/pro-labor stimuli. The present study examined this issue by assessing the effects of forskolin on P4/PR anti-
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inflammatory activity and pSer344/345-PRA generation in an immortalized myometrial cell line model. We found that forskolin increased P4/PR anti-inflammatory actions, in part, by inhibiting
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the generation of pSer344/345-PRA via increased expression of dual specificity protein phosphatase 1 (DUSP1), that encodes a phosphatase that inactivates MAPKs. In addition, we found that the generation of pSer344/345-PRA in hTERT-HMA/B cells was catalyzed by stressactivated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in response to IL-1ß. Taken together the data suggest that the cAMP and P4/PR signaling pathways act synergistically to inhibit the response of myometrial cells to pro-inflammatory stimuli and that this is in part mediated by DUSP1 inhibition of specific MAPKs (especially SAPK/JNK) in myometrial cells. This may be a key mechanism for the maintenance of uterine quiescence for most of pregnancy Page 4
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that could be exploited therapeutically to prevent preterm labor and its progression to preterm birth.
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METHODS Cell culture: Studies were performed in a telomerase-immortalized human myometrial cell line, hTERT-HMA/B (7,25), that contains stably incorporated and independently inducible transgenes
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encoding PR-A in response to doxycycline (DOX) and PR-B in response to diacylhydrazine (DAH) (7). The PR-A and PR-B transgenes are silent in the absence of exposure to DOX and
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DAH. Cell cultures were maintained at 37°C in a 5% CO2 humidified incubator in Dulbecco's modified eagle medium (DMEM)/Ham's F12 (1:1) supplemented with 5% charcoal-stripped fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1 mg/ml geneticin and 2 mM L-glutamine (Life Technologies, Grand Island, NY).
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Transient transfection: hTERT-HMA/B cells at 80-90% confluence were harvested by trypsinization, pooled into an electroporation cuvette at a concentration of 1 x 106 cells/100µL transfection solution (Mammalian Smooth Muscle cell transfection solution: Lonza, Walkersville,
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MD) containing the DNA or RNA to be transfected, and subjected to electroporation (program A033 in the Amaxa Nucleofector; Lonza). The cells were then re-plated in standard culture media
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(above) and allowed to stabilize for 16h before exposure to experimental conditions.
Immunoblotting: Abundance of specific proteins in hTERT-HMA/B total cell lysate was measured by immunoblotting. After experimental treatments, hTERT-HMA/B cells were washed in
ice-cold
PBS,
collected
by
scraping,
pelleted
by
centrifugation
and
lysed
in
radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma; St Louis MO) as previously described (10). The lysates were then centrifuged at 16000×g for 10min at 4˚C. Supernatants were assayed for protein content using the Page 5
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bicinchoninic acid protein assay (Thermo Fisher Scientific). Equal amounts of lysate protein were diluted in gel loading buffer [375mM Tris-HCl, 6% sodium dodecyl sulfate (SDS), 48% glycerol, 9% β-mercaptoethanol and 0.03% bromophenol blue, pH 6.8], heated at 100°C for 5
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min and subjected to denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on pre-cast 4-20% Tris-glycine polyacrylamide gels using the Novex electrophoresis system (Life Technologies). Proteins were then electro-transferred to a
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polyvinylidene difluoride membrane (Millipore). For imumunodetection, membranes were first incubated in blocking buffer [5% nonfat milk in Tris-buffered saline containing 0.1% tween-20
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(TBST)] at room temperature for 1h and then with primary antibodies (Table 1) diluted in 5% nonfat milk/TBST overnight at 4˚C. The following day membranes were washed three times with TBST and incubated at room temperature for 1h with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit diluted in 5% nonfat milk/TBST. After three washes with TBST,
immunoreactive
proteins
were
detected
by
chemiluminescence
(HyGlo
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chemiluminescent reagent; Denville Scientific) and quantified by digital fluorescent densitometry (FluorChem E processor; ProteinSimple, San Jose , CA).
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mRNA abundance: Total RNA was isolated from cells using a commercial kit (Total RNA Isolation/NucleoSpin RNA II kit; Macherey-Nagel, Bethlehem, PA), treated with DNase (DNA-
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free; Life Technologies) and quantified by absorbance at 260nM. 300ng of total RNA was then reverse-transcribed with random hexamers using Superscript II Random Prime Synthesis kit for RT-PCR (Thermo Fisher Scientific, Grand Island, NY). Aliquots of cDNA were then used as template for quantitative reverse transcription polymerase chain reaction (qRT-PCR) with primers (Table 2) for transcripts of interest (designed using the PrimerExpress application; Applied Biosystems) on the StepOnePlus real-time PCR system (Applied Biosystems) using SYBR Green (Thermo Fisher Scientific) as the fluorescent detector to measure amplicon abundance in real-time. Abundance of specific mRNA relative to GAPDH mRNA was calculated Page 6
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using the ∆CT method [relative mRNA abundance = 2 − (CT gene of interest − CT GAPDH)]. Assays were validated for primer specificity and optimized to ensure the equivalent PCR efficiency for each
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primer pair.
P4/PR transcriptional activity via the progesterone response element: To assess P4/PR transcriptional activity, hTERT-HMA/B cells were transiently transfected with DNA containing the
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firefly luciferase (LUCFF) open reading frame controlled by a promoter comprised of tandem canonical progesterone response elements (PREs) (PRE2-LUCFF) and DNA containing the
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renilla LUC (LUCRN) controlled by the constitutively active cytomegalovirus (CMV) promoter (CMV-LUCRN). LUCFF and LUCRN activity were measured in cell lysates using the DualLuciferase reporter assay system (Promega, Madison WI) on a GloMax 20/20 luminometer (Promega).
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Statistical analyses: A minimum of n=3 replicates were used for each experimental condition and experiments were repeated in at least 3 time-separated studies. Normally distributed data (determined by Kolmogorov-Smirnov test) were compared using Student t test and analysis of
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variance (ANOVA) with post hoc Dunnett test. Non-normally distributed data were compared by Wilcoxon rank-sum test or Mann Whitney U test. Differences were considered statistically
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significant when the P value < 0.05.
RESULTS
Anti-inflammatory effects of P4/PR and forskolin: hTERT-HMA/B cells were maintained in the basal state (negligible PR levels and no PRE2-LUCFF response to P4) or induced to express equivalent amounts of PR-A and PR-B and then treated with P4 (100nM; Sigma) or vehicle [dimethyl sulfoxide (DMSO)] for 24h. Media was then changed and the cells were exposed to IL1ß (1ng/mL; Cell Signaling) in the presence and absence of P4 (replenished) and/or forskolin Page 7
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(10 µM; Sigma) for a further 24h. Total RNA and whole cell lysate were then prepared and used, respectively, to measure relative abundance of IL-8 mRNA by qRT-PCR, and protein by immunoblotting. As expected, IL-1ß increased the amount of IL-8 protein (Figure 1A) and mRNA
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(Figure 1B) in hTERT-HMA/B cells. This effect was inhibited by P4 only in cells induced to express PRs. Forskolin also inhibited IL-1ß-induced IL8 expression. The combination of P4 and forskolin produced a greater inhibition of IL-1ß-induced IL8 expression than either agent alone
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(Figures 1A and 1B). Inhibition of IL-1ß-induced IL8 expression by forskolin was dosedependent, mimicked by 8-bromoadenosine-cAMP (8-Br-cAMP; Sigma), a cAMP mimetic that is
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resistant to phosphodiesterase degradation (Figure 1C), and inhibited by H89 (10µM; Sigma), a PKA inhibitor, suggesting that anti-inflammatory actions of forskolin were mediated by activation of PKA by cAMP (Figure 1D).
Effect of forskolin and P4/PR on expression of labor-associated genes: The effect of
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forskolin on basal and P4- and IL-1ß-induced expression of genes encoding labor-associated inflammatory mediators and contraction-associated proteins (CAPs) was examined (Figure 2). For this experiment, hTERT-HMA/B cells were conditioned to express PR-A and PR-B (1:1) and
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exposed to P4 (100nM) or vehicle for 24h. The cells were then exposed to IL-1ß (1 ng/mL) in the presence of vehicle or increasing concentrations of forskolin (0.1-100 µM). Total RNA was
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then isolated and subjected to qRT-PCR to measure the extent of expression (assessed by the relative amount of cognate mRNAs) of genes encoding the oxytocin receptor (OXTR), the PGF2α receptor (PTGFR), connexin-43 (GJA1), IL-8 (IL8), prostaglandin-endoperoxide synthase-2 (PTGS2) and IL-6 (IL6) (Figure 2). Forskolin and P4/PR suppressed OXTR, GJA1, IL8, PTGS2 and IL6 expression in a forskolin concentration-dependent manner. P4/PR and forskolin had synergistic effects on the inhibition of IL-1ß-induced expression of IL8, PTGS2 and IL6 expression. There was no apparent synergistic effect of P4/PR and forskolin on OXTR and GJA1 expression, and neither agent affected expression of PTGFR. Page 8
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Effect of forskolin on P4/PR transcriptional activity: To examine the mechanism for synergy between P4/PR and cAMP in myometrial cells, the effect of forskolin on PR transcriptional
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activity was measured. To do this, hTERT-HMA/B cells were transiently co-transfected with PRE2-LUCFF and CMV-LUCRN as previously described (10). Cells were induced to express only PR-A, only PR-B, both PRs or left un-induced and exposed to P4 (100 nM) or vehicle for 24h.
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Media was then replenished with P4 or vehicle, and the cells exposed to forskolin (10 µM) for an additional 24h. LUCFF and LUCRN activity was then measured in whole cell lysates. As expected,
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P4 increased PRE2-LUCFF expression in cells induced to express PR-B but not in cells expressing only PR-A and cells lacking PRs (Figure 3A). Typical of PR-A trans-repressive activity, PR-A inhibited P4-induced PRE2-LUCFF expression mediated by PR-B. Forskolin did not alter P4/PR-induced expression of PRE2-LUCFF or PR-A trans-repression of PR-B at the PRE2LUCFF promoter.
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The effect of forskolin on P4/PR transcriptional activity was also examined using an endogenous P4/PR-responsive gene, FKBP5, whose promoter contains multiple PREs. We previously found that in hTERT-HMA/B cells treatment with P4 via PR-B but not PR-A induces
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FKBP5 expression (26). Expression of FKBP5 was not affected by forskolin (Figure 3B). Interestingly, unlike PRE2-LUCFF, PR-A did not trans-repress the activity of P4/PR-B-induced
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FKBP5 expression. These data are consistent with our previous studies showing that transrepressive activity of PR-A at the FKBP5 promoter is absent under basal conditions in hTERTHMA/B cells (26).
Effect of forskolin on pSer344/345-PRA generation: The hypothesis that cAMP increases myometrial cell P4/PR anti-inflammatory activity by inhibiting the generation of pSer344/345-PRA was tested. Cells were induced to express PR-A and PR-B and exposed to P4 or vehicle for 24h. Media and test agents were then replenished and some cultures were exposed to IL-1ß (1 Page 9
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ng/mL) and/or forskolin (10 µM) for a further 24h. Whole cell lysate was then prepared and used to determine the abundance of PR-A/B (total PRs), pSer344/345-PRs, IL-8 and GAPDH by immunoblotting (Figure 4). As expected, P4 increased pSer344/345-PRA and this was augmented
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by IL-1ß. Forskolin inhibited P4 and IL-1ß induced generation of pSer344/345-PRA.
Role of MAPKs in pSer344/345-PRA generation: The mechanism underlying the generation of pSer344/345-PRA in response to P4 and IL-1ß and its inhibition by forskolin was further explored
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by determining the role of MAPKs in catalyzing the generation of pSer344/345-PRA in hTERT-
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HMA/B cells. Generation of pSer344/345-PRA was abolished by the pan-RAF-kinase inhibitor LY3009120 (10 µM) that inhibits the activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p38 kinase, and stress-activated protein kinase/c-Jun N-terminal Kinase (SAPK/JNK), suggesting that these MAPKs catalyze pSer344/345-PRA formation (Figure 5A). The effect of forskolin on ERK1/2, p38 and SAPK/JNK content and activation (indicated by
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the level of respective phospho-forms) in hTERT-HMA/B cells treated to express PR-A and PR-B and then exposed to P4 (100nM), IL-1ß (1ng/mL) and various concentrations of forskolin was determined. Forskolin inhibited P4- and P4/IL-1ß-induced pSer344/345-PRA in a concentration-
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dependent manner (Figure 5B). The decrease in pSer344/345-PRA was associated with a parallel decrease in pThr202/Tyr204-ERK1/2 and pThr183/Tyr185-SAPK/JNK but not pThr180/Tyr182-p38.
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Further analyses were performed using U0126 (20µM) and SP600125 (100µM), small molecule inhibitors of ERK1/2 and SAPK/JNK, respectively. For these experiments, hTERT-HMA/B cells were exposed to U0126, SP600125 or vehicle for 1h and then to P4 for a further 1h. The 2h (vs 24h) time of exposure was chosen to avoid toxic effects of long-term (24h) exposure to U0126 and SP600125. Generation of pSer344/345-PRA and -PRB was inhibited by the SAPK/JNK inhibitor, SP600125, but not by the ERK1/2 inhibitor, U0126 that specifically decreased the level of phospho-ERK1/2 (Figure 5C). We previously showed that P4 induces the generation of pSer344/345-PRA and -PRB during the first 1-2h of exposure to P4 (9). Page 10
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Mechanism for cAMP inhibition of MAPK activity: Studies in other cell types suggest that cAMP inhibits MAPK activation by inducing the expression of DUSP1 that encodes dual specific
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protease-1 (DUSP1), a phosphatase that inactivates (by de-phosphorylation) MAPKs (27-30). It is also possible that anti-inflammatory actions of P4/PR are mediated by increased DUSP1 expression. We therefore determined whether P4/PR and forskolin affect DUSP1 expression
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(based on DUSP1 mRNA levels measured by qRT-PCR) in hTERT-HMA/B cells treated as described in Figure 2. P4/PR and forskolin increased DUSP1 expression. P4/PR caused a leftand up-shift in the forskolin concentration-response (Figure 6A).
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To determine whether DUSP1 mediates the inhibitory effect of forskolin on pSer344/345-PRA generation, its levels were decreased by RNA interference (RNAi) in hTERT-HMA/B cells by transfecting the cells with short interfering RNAs (Stealth siRNAs; Stealth ID:VHS40581 and VHS40583; Thermo Fisher) targeting the open reading frame of DUSP1 or with scrambled
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siRNAs (negative control). The cells were then induced to express PR-A and PR-B and exposed to P4 (100 nM), IL-1ß (1 ng/mL) and forskolin (10 µM) for 24h. Whole cell lysate was then prepared and subjected to immunoblotting to determine the levels of pSer344/345-PRA and
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pSer359-DUSP1 (Figure 6B). DUSP1 knock down by RNAi decreased pSer359-DUSP1. The decrease in DUSP1 was associated with increased pSer344/345-PRA in P4 treated cells and
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attenuated the inhibitory effects of forskolin on pSer344/345-PRA.
DISCUSSION
It is well-established that the cAMP/PKA signaling cascade promotes myometrial cell relaxation by decreasing intracellular free Ca2+ and preventing phosphorylation/activation of the myosin light chain (31-33). Consequently, this signaling pathway is thought to play a central role in promoting relaxation of the myometrium necessary for pregnancy. Indeed, various hormones acting via Gαs-linked receptor that activate adenylyal cyclase, have been implicated as
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contributing, via cAMP, to myometrial quiescence during pregnancy. However, cAMP signaling alone is not sufficient to maintain myometrial relaxation because it cannot compensate for the onset of labor triggered by P4/PR withdrawal. Moreover, the management of preterm labor with
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ß2-agonists has severe potentially life-threatening side effects on the maternal cardiovascular system and has limited tocolytic effectiveness due to pharmacologic down-regulation of adrenergic receptors in myometrial cells. Thus, although cAMP signaling promotes myometrial
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cell relaxation, this pathway is not sufficient to block labor in the context of P4 withdrawal. Instead, several studies suggest that cAMP signaling in myometrial cells functions in concert
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with P4/PR signaling to promote uterine quiescence. Chen et al.(8) reported that cAMP (induced by forskolin) increased the anti-inflammatory actions of P4 [likely acting via the glucocorticoid receptor (GR) in their model system] in primary cultures of term myometrial cells, and studies in breast cancer cells show that cAMP qualitatively and qualitatively modulates PR transcriptional activity (23,34). This evidence supports a functional cross-talk between cAMP and P4/PR
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signaling in myometrial cells that is distinct from effects of cAMP/PKA on the contractile apparatus and may be critical for the maintenance of pregnancy by modulating PR transcriptional activity. The present studies tested this hypothesis by focusing on the effect of
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cAMP (induced by forskolin) on the capacity for P4/PR to exert anti-inflammatory actions in myometrial cells and how its effects may be mediated by PR phosphorylation. Studies were
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performed in a human myometrial cell line, hTERT-HMA/B, genetically modified to allow experimental manipulation of PR-A and PR-B levels. IL-1ß was used as a prototypical pro-labor inflammatory stimulus, and expression of IL8 was measured as a prototypical readout for IL-1ßinduced pro-inflammatory/pro-labor action. IL-8 is a potent chemokine that promotes tissue-level inflammation and IL8 expression in myometrial cells increases in association with the onset of labor (34-36). cAMP signaling was induced by exposing cells to forskolin, an activator of adenylyl cyclase that increases intracellular levels of cAMP.
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We found that P4/PR and forskolin inhibited IL-1ß-induced IL8 expression via independent, yet functionally linked and synergistic, pathways (Figure 1). The combination of P4/PR and cAMP signaling promoted a gene expression profile consistent with myometrial relaxation and
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uterine quiescence based on decrease in expression of OXTR, GJA1, PTGS2, IL8 and IL6 (Figure 2). The data support the hypothesis that cAMP and P4/PR signaling in myometrial cells synergize to inhibit the response of myometrial cells to pro-labor/pro-inflammatory stimuli and
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affect expression of specific genes to promote uterine quiescence. This effect, coupled with cAMP-induced PKA-mediated inhibition of myometrial cell contraction may be critical for
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maintaining uterine quiescence during pregnancy.
We examined the mechanism by which forskolin increased the capacity for P4/PR to inhibit responsiveness to IL-1ß. In this regard, our studies extended those of Chen et al. (8) who proposed that, in primary myometrial cell cultures forskolin increased the capacity for P4 to inhibit IL-1ß-induced PTGS2 expression by increasing PR transcriptional activity. They found
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that forskolin increased the capacity for P4 to induced expression of FKBP5 and 11ßHSD1 expression, and PRE-LUC expression in cells transfected to over-express PR-B. The FKBP5 promoter contains multiple canonical PRE/glucocorticoid receptor response elements and its
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expression is induced by PR and GR. In this context, it is notable that P4 actions reported by Chen et al. were likely mediated via the GR, especially as the concentration of P4 used to exert
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an anti-inflammatory effects and drive gene expression was 100 µM, which is high (we used 100 nM) and likely sufficient to activate the GR. We therefore, re-examined the capacity for forskolin to affect P4/PR transcriptional activity using our hTERT-HMA/B cell model in which levels of PRA and PR-B were experimentally controlled. We found that, as expected, P4 induced PRE2LUCFF and FKBP5 expression and that this occurred only in cells induced to express PR-B, and that P4/PR-A trans-repressed P4/PRB-induced PRE2-LUCFF expression. Importantly, forskolin did not affect the capacity for P4/PR-B to induce expression of PRE2-LUCFF and FKBP5 or the capacity for PR-A to trans-repress PR-B activity at the PRE2-LUCFF promoter (Figure 3). Thus, Page 13
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our data suggest that effects of forskolin on P4/PR anti-inflammatory activity were not mediated by changes in canonical PR/PRE transcriptional activity. Recent studies suggest that P4/PR transcriptional activity in myometrial cells can occur by a
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non-canonical pathway whereby the PRs interact directly with other transcription factors (37-39), and that this is controlled via site-specific serine phosphorylation (37). Importantly, we previously found that pSer344/345-PRA inhibits net P4/PR anti-inflammatory activity in hTERT-
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HMA/B cells and that levels of pSer344/345-PRA were higher in laboring compared to quiescent term myometrium (9). Importantly, we found that in explant cultures of term myometrium and in
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hTERT-HMA/B cells, the generation of pSer344/345-PRA was P4-dependent and induced by IL-1ß. Those data led to the hypothesis that human parturition involves the induction of protein kinase activity in myometrial cells in response to pro-inflammatory stimuli that catalyzes pSer344/345-PRA generation, and that pSer344/345-PRA causes functional withdrawal of P4/PR anti-inflammatory activity leading to tissue level inflammation. Interestingly, our data suggest that inhibition of
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pSer344/345-PRA generation in myometrial cells is a hallmark of uterine quiescence, and in this context our finding that forskolin inhibited the generation of pSer344/345-PRA in hTERT-HMA/B cells further supports the notion that cAMP signaling promotes the quiescent phenotype in
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myometrial cells. Our finding that forskolin inhibited expression of OXTR, GJA1, IL8, IL6 and PTGS2 expression further supports this hypothesis and that synergism between the P4/PR and
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cAMP/PKA pathways extends beyond anti-inflammatory actions to affect a specific cassette of contraction-related and inflammatory genes. We found that the generation of pSer344/345-PRA in hTERT-HMA/B cells was induced by the SAPK/JNK kinase. This is consistent with the hypothesis that human parturition involves inflammation-induced functional P4/PR withdrawal mediated by the generation of pSer344/345PRA in response to stress-associated pro-inflammatory stimuli. Our finding that forskolin inhibited pSer344/345-PRA generation suggests that cAMP decreases SAPK/JNK activity in myometrial cells. One mechanism for this effect could be via cAMP-induced expression of Page 14
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DUSP1 (40). Previous studies have shown that P4 increases DUSP1 expression in myometrial cells, and that knockdown of DUSP1 abolished the capacity for P4 to inhibit IL-1ß-induced c-Jun activation and COX2 expression (38,41). Other studies showed that treatment of mice with
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phosphodiesterase inhibitors that elevate the level of cAMP, increased DUSP1 expression in different cell types, and that anti-inflammatory effects of phosphodiesterase inhibitors were significantly lower in DUSP1 knockout mice (28,42-44). We found that in hTERT-HMA/B cells P4/PR and forskolin increased DUSP1 expression in a cooperative manner, and that inhibition
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of DUSP1 by RNAi increased P4-induced pSer344/345-PRA and attenuated the inhibitory effects of forskolin on pSer344/345-PRA generation (Figure 6). These findings suggest that cAMP inhibits
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pSer344/345-PRA generation in myometrial cells, at least in part, by increasing DUSP1 expression and/or activity. Thus, consistent with the conclusions of Lei et al. (38), DUSP1 may be a key mediator of the cross-talk between cAMP and P4/PR signaling in myometrial cells and suppresses responsiveness to pro-inflammatory stimuli that function via MAPKs, and especially
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SAPK/JNK. This synergism may be critical for the maintenance of uterine quiescence for most of pregnancy. Further studies of this cross-talk may reveal novel therapeutic strategies to
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promote uterine quiescence and prevent preterm labor.
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Acknowledgments
We thank Lora J Mesiano for administra ive and editorial assistance. This work was funded by grants to SM by the March of Dimes Prematurity Center, Ohio Collaborative, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (HD069819).
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FIGURE 1
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FIGURE 2
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TABLE 1: Antibodies used for immunoblotting Manufacturer
Catalogue No.
Dilution
PR-A/B
Dako
M3568
1:1000
pSer344/345-PRA/B
Cell Signaling Technology
12783
1:1000
pSer359-DUSP1
Cell Signaling Technology
2857
1:1000
ERK1/2
Cell Signaling Technology
4696
1:1000
pThr202/Tyr204-ERK1/2
Cell Signaling Technology
9101
1:1000
p38
Cell Signaling Technology
9212
1:1000
pThr180/Tyr182-p38
Cell Signaling Technology
9211
1:1000
SAPK/JNK
Cell Signaling Technology
9252
1:1000
pThr183/Tyr185-SAPK/JNK
Cell Signaling Technology
9255
1:1000
Mouse IgG
Cell Signaling Technology
7076
1:3000
Rabbit IgG
Cell Signaling Technology
7074
1:3000
IL-8
R&D systems
MAB208
1:1000
SantaCruz Biotechnology
32233
1: 100000
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GAPDH
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Protein Target
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TABLE 2: Primer sequences for qRT-PCR Forward Primer
Reverse Primer
IL8
TGGCAGCCTTCCTGATTTCT
TTA GCA CTC CTT GGC AAA ACT G
PTGS2
ATGTTCCACCCGCAGTACAGA
CAGCATAAAGCGTTTGCGGTA
FKBP5
ATGCCATTTACTGTGCAAACCAG
AAGAGAGTTGCATTCGAGGGAA
GAPDH
TTGCCATCAATGACCCCTTCA
CGCCCCACTTGATTTTGGA
GJA1
TGG CCT TCT TGC TGA TCC A
TTT GCA AGT GTA AAC AGC ACT CAA
OXTR
CTG GAC GCC TTT CTT CTT CGT
GAA GGC CGA GGC TTC CTT
PTGFR
AGCAGTTTCAAAACTCTACCATGG
TGCCAATATTCTTTGCACCTATCA
IL6
TAGCCGCCCCACACAGA
CCGTCGAGGATGTACCGAAT
DUSP1
GCCACCATCTGCCTTGCTTA
TTCACAAACTCAAAGGCCTCG
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Gene
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FIGURE LEGENDS
Figure 1: Anti-inflammatory effects of P4/PR and forskolin. Effect of P4/PR, forskolin and 8Br-cAMP on IL-1ß-induced IL8 expression in hTERT-HMA/B cells. A: Immunoblot analyses of
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PR-A/B (total PRs), GAPDH and IL-8 in whole cell lysate from hTERT-HMA/B cells under basal conditions or induced to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) and forskolin (10 µM) for 24h. B: Relative
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abundance of IL8 mRNA measured by qRT-PCR in hTERT-HMA/B cells treated as described for A. C: Immunoblot analysis for PR-A/B (total PRs), GAPDH and IL-8 in whole cell lysate from
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hTERT-HMA/B cells treated to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) and varying concentrations of forskolin (upper) and 8-Br-cAMP (lower). D: Relative abundance of IL8 mRNA measured by qRT-PCR in hTERT-HMA/B cells treated to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and/or IL-1ß (1 ng/mL) in the presence of H89 (10 µM; Sigma).
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Immunoblot data are representative of 3 time-separated experiments. Different gels are separated by white space. mRNA data are mean ± SE of n=3 and representative of 3 time-
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separated experiments. * P<0.05.
Figure 2: Effect of forskolin and P4/PR on expression of labor-associated genes. Relative
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abundance of RNAs (assayed by qRT-PCR) encoding OXTR, GJA1, PTGFR, IL-8, PTGS2, and IL-6 in total RNA from hTERT-HMA/B cells induced to express PR-A (DOX: 100 ng/mL) and PRB (DAH: 400 nM) and then exposed to P4 or vehicle for 24h and then exposed to IL-1ß (1 ng/ml) and increasing concentrations of forskolin (0-100 µM) for a further 24h. Mean ± SE; n=3.
Figure 3: Effect of forskolin on P4/PR transcriptional activity. A: Ratio of firefly LUC activity (from the PRE-LUC reporter) to renilla LUC activity (from CMV-LUC reporter) in hTERT-HMA/B
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cells under basal conditions or induced to express PR-A and/or PR-B, transiently co-transfected with PRE-LUC and CMV-LUC DNA and treated with vehicle or DOX (100 ng/mL) and/or DAH (400 nM) for 24h to induce PR-A and/or PR-B expression. Cells were then exposed to forskolin
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(10µM) and/or P4 (100nM) or vehicle for a further 24h. Cell lysate was then assayed for firefly and renilla luciferase activity. Mean ± SE n=3. B: Mean ± SE (n=3) relative abundance (measured by qRT-PCR) of mRNA encoding FKBP5 in hTERT-HMA/B cells treated as described
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to express PR-A and/or PR-B and then treated with P4 and/or forskolin. Mean ± SE (n=3).
Figure 4: Effect of forskolin on pSer344/345-PRA generation. Immunoblot analysis of PR-A/B
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(total PRs), pSer344/345-PR and IL-8 in hTERT-HMA/B cells conditioned to express PR-A and PR-B and then treated with vehicle or P4 (100 nM). Cells were then exposed to IL-1ß (1 ng/ml) and/or forskolin (10 µM) (with P4 replacement) for additional 24h. Representative of 3 identical time-
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Figure 5: Role of MAPKs in pSer344/345-PRA generation: hTERT-HMA/B cells were induced to express PR-A and PR-B and exposed to test agents to determine the role of MAPKs in
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pSer344/345-PRA generation. A: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and GAPDH in lysate from cells exposed to RAF-kinase inhibitor, LY30019120 (10 µM; inhibits the
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activation of all MAPKs) for 1h and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) for additional 24h. B: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and total- and phospho-MAPKs: ERK1/2; p38 and SAPK/JNK in lysate from cells exposed to P4 (100 nM) for 24h and then challenged with IL-1ß (1 ng/mL) and increasing concentrations of forskolin (0.1 100µM) for additional 24h. C: Left: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR, phospho-ERK1/2 and GAPDH in lysate from cells treated with U0126 (20 µM: inhibits generation of active phospho-ERK1/2 by inhibiting MEK1/2 kinase) and SP600125 (100 µM: inhibits SAPK/JNK activity) for 1h and then exposed to vehicle or P4 (100nM) for a further 1h. Page 25
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Right: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and GAPDH in lysate from cells treated with vehicle or increasing concentrations of SP600125 (25-100 µM) for 1h and then exposed to vehicle or P4 (100nM) for a further 1h. Different gels are separated by white space.
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Data are representative of 3 identical time-separated experiments.
Figure 6: Mechanism for forskolin inhibition of MAPK activity. A: Relative abundance of
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DUSP1 mRNA measured by qRT-PCR in hTERT-HMA/B treated with P4 (100 nM) and increasing concentrations of forskolin (0.1 - 100µM). Mean ± SE; n=3. B: Immunoblot analysis of
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PR-A/B (total PRs), pSer344/345-PR, GAPDH and phospho-DUSP1 in lysate from cells transiently transfected with siRNAs targeting the DUSP1 open reading frame or siRNAs with scrambled sequence and then treated with P4 (100 nM) and forskolin (10 µM) for 24h. Representative of
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n=3 time-separated experiments.
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Progesterone (P4) and cAMP synergistically inhibit myometrial cells response to IL-1ß
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cAMP and P4/PR inhibit expression genes encoding contraction-associated
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factors. cAMP inhibits the phosphorylation of PR-A and induces the expression of DUSP1
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DUSP1 inhibits phosphorylation of PR-A
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S0303-7207(18)30240-5
DOI:
10.1016/j.mce.2018.08.005
Reference:
MCE 10283
To appear in:
Molecular and Cellular Endocrinology
Received Date: 12 June 2018 Revised Date:
13 August 2018
Accepted Date: 13 August 2018
Please cite this article as: Amini, P., Wilson, R., Koeblitz, W., Wang, J., Tan, H., Yi, L., Stanfield, Z., Romani, A., Malemud, C., Mesiano, S., Mechanism by which progesterone and cAMP synergize to maintain uterine quiescence during pregnancy, Molecular and Cellular Endocrinology (2018), doi: 10.1016/j.mce.2018.08.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Mechanism by which Progesterone and cAMP Synergize to Maintain Uterine Quiescence during Pregnancy
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Peyvand Amini3, Rachel Wilson1, William Koeblitz1, Junye Wang1, Huiqing Tan1, Lijuan Yi1, Zachary Stanfield5, Andrea Romani3, Charles Malemud4, Sam Mesiano1,2 1
Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH
2
Department of Obstetrics and Gynecology, University Hospitals Cleveland Medical Center,
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Cleveland OH 3
Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH
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Department of Medicine, Case Western Reserve University, Cleveland, OH
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Systems Biology and Bioinformatics, Case Western Reserve University, Cleveland, OH
Abbreviated title: Synergism between progesterone and cAMP
Key terms: progesterone receptor, cyclic AMP, pregnancy, inflammation, myometrium
Sam Mesiano, PhD
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Corresponding author:
Department of Reproductive Biology
11100 Euclid Ave, Cleveland, OH 44106
Fax: 216-844-7095
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Phone: 216-844-1553
Email: [email protected]
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Grants supporting this study: The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (HD069819). March of Dimes Prematurity Center, Ohio Collaborative.
Disclosure statement: The authors have nothing to disclose
Short title: Anti-inflammatory actions of progesterone and cAMP Page 1
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ABSTRACT Progesterone (P4) acting through the P4 receptor (PR) isoforms, PR-A and PR-B, promotes uterine quiescence for most of pregnancy, in part, by inhibiting the response of myometrial cells
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to pro-labor inflammatory stimuli. This anti-inflammatory effect is inhibited by phosphorylation of PR-A at serine-344 and -345 (pSer344/345-PRA). Activation of the cyclic adenosine monophosphate (cAMP) signaling pathway also promotes uterine quiescence and myometrial relaxation. This study examined the cross-talk between P4/PR and cAMP signaling to exert anti-
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inflammatory actions and control pSer344/345-PRA generation in myometrial cells. In the hTERT-
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HMA/B immortalized human myometrial cell line P4 inhibited responsiveness to interleukin (IL)1ß and this anti-inflammatory effect was increased by forskolin (increases cAMP) and 8-BrcAMP in a concentration-dependent and synergistic manner that was mediated by activation of protein kinase A (PKA). Forskolin also inhibited the generation of pSer344/345-PRA and expression of key contraction-associated genes in a concentration-dependent manner.
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Generation of pSer344/345-PRA was catalyzed by stress-activated protein kinase/c-Jun NH2terminal kinase (SAPK/JNK). Forskolin inhibited pSer344/345-PRA generation, in part, by increasing the expression of dual specificity protein phosphatase 1 (DUSP1), a phosphatase
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that inactivates mitogen-activated protein kinases (MAPKs) including SAPK/JNK. P4/PR and forskolin increased DUSP1 expression. The data suggest that P4/PR promotes uterine
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quiescence via cross-talk and synergy with cAMP/PKA signaling in myometrial cells via DUSP1mediated inhibition of SAPK/JNK activation.
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INTRODUCTION Parturition (the process of birth) involves transition of the uterus from the quiescent to the laboring state wherein the myometrium (the smooth muscle of the uterine wall) produces phasic
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and forceful contractions to become the engine for birth. In women, a major trigger for parturition is tissue-level inflammation in the myometrium that involves the infiltration and activation of immune cells and the local production of pro-inflammatory cytokines, especially interleukin (IL)-
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1ß (IL-1ß) and prostaglandins (PGs) (1-5). For most of pregnancy the steroid hormone progesterone (P4), via its interaction with the nuclear P4 receptor (PR) isoforms, PR-A and PR-
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B, in myometrial cells blocks labor, in part, by inhibiting responsiveness to pro-labor/proinflammatory stimuli (i.e., an anti-inflammatory effect), thus preventing tissue-level inflammation (6-10). In all viviparous species, parturition is induced by the withdrawal of P4/PR signaling and in women this is thought to be mediated by the functional loss of P4/PR anti-inflammatory activity via changes in PR transcriptional activity (11,12). Our previous studies suggest that
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phosphorylation of PR-A at serine residues -344 and -345 (pSer344/345-PRA; numbers relative to amino acid 1 in PR-B) in myometrial cells causes functional P4/PR withdrawal (9). The level of pSer344/345-PRA in term myometrium was markedly higher in laboring compared with non-
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laboring tissue, and that generation of pSer344/345-PRA in myometrial cells was P4-dependent and induced by IL-1ß. Importantly, we also found that the capacity for P4/PR to exert anti-
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inflammatory activity in myometrial cells was inhibited by pSer344/345-PRA. The data suggest that human parturition involves pSer344/345-PRA-mediated functional P4/PR withdrawal and that prevention of pSer344/345-PRA generation in myometrial cells is important to maintain uterine quiescence for most of pregnancy. The 3',5'-cyclic adenosine monophosphate (cAMP) intracellular signaling cascade is a major inhibitor of smooth muscle contractility and is thought to play a key role in maintaining myometrial quiescence during pregnancy (13,14). cAMP is produced by the adenylyl cyclase enzyme which is activated by hormones that bind Gαs-coupled transmembrane receptors. In Page 3
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most cells cAMP functions as a second messenger that activates protein kinase-A (PKA), a multifunctional kinase that phosphorylates downstream targets to induce a variety of cellular response (15). In smooth muscle cells PKA suppresses contractility by inhibiting myosin light
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chain kinase activity and decreasing the intracellular concentration of free Ca2+ (16-21). cAMP may also contribute to the maintenance of pregnancy by exerting anti-inflammatory activity in myometrial cells, and by boosting P4/PR transcriptional activity (8). Studies in airway smooth
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muscle cells show that cAMP signaling inhibits response to pro-inflammatory stimuli by suppressing the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-
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κB) and mitogen-activate protein kinases (MAPKs) (22). In breast cancer cell lines, cAMP affects P4/PR signaling in a quantitative and qualitative manner (23,24), and in myometrial cells derived from term uterus, forskolin, a bicyclic diterpene that increase intracellular cAMP by stimulating adenylyl cyclase, increases the anti-inflammatory activity of P4 (8). Thus, the functional interaction between P4/PR and cAMP signaling in myometrial cells may be critical for
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the maintenance of uterine quiescence during pregnancy by promoting myometrial cell relaxation and refractoriness to pro-inflammatory/pro-labor stimuli. The present study examined this issue by assessing the effects of forskolin on P4/PR anti-
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inflammatory activity and pSer344/345-PRA generation in an immortalized myometrial cell line model. We found that forskolin increased P4/PR anti-inflammatory actions, in part, by inhibiting
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the generation of pSer344/345-PRA via increased expression of dual specificity protein phosphatase 1 (DUSP1), that encodes a phosphatase that inactivates MAPKs. In addition, we found that the generation of pSer344/345-PRA in hTERT-HMA/B cells was catalyzed by stressactivated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in response to IL-1ß. Taken together the data suggest that the cAMP and P4/PR signaling pathways act synergistically to inhibit the response of myometrial cells to pro-inflammatory stimuli and that this is in part mediated by DUSP1 inhibition of specific MAPKs (especially SAPK/JNK) in myometrial cells. This may be a key mechanism for the maintenance of uterine quiescence for most of pregnancy Page 4
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that could be exploited therapeutically to prevent preterm labor and its progression to preterm birth.
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METHODS Cell culture: Studies were performed in a telomerase-immortalized human myometrial cell line, hTERT-HMA/B (7,25), that contains stably incorporated and independently inducible transgenes
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encoding PR-A in response to doxycycline (DOX) and PR-B in response to diacylhydrazine (DAH) (7). The PR-A and PR-B transgenes are silent in the absence of exposure to DOX and
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DAH. Cell cultures were maintained at 37°C in a 5% CO2 humidified incubator in Dulbecco's modified eagle medium (DMEM)/Ham's F12 (1:1) supplemented with 5% charcoal-stripped fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1 mg/ml geneticin and 2 mM L-glutamine (Life Technologies, Grand Island, NY).
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Transient transfection: hTERT-HMA/B cells at 80-90% confluence were harvested by trypsinization, pooled into an electroporation cuvette at a concentration of 1 x 106 cells/100µL transfection solution (Mammalian Smooth Muscle cell transfection solution: Lonza, Walkersville,
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MD) containing the DNA or RNA to be transfected, and subjected to electroporation (program A033 in the Amaxa Nucleofector; Lonza). The cells were then re-plated in standard culture media
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(above) and allowed to stabilize for 16h before exposure to experimental conditions.
Immunoblotting: Abundance of specific proteins in hTERT-HMA/B total cell lysate was measured by immunoblotting. After experimental treatments, hTERT-HMA/B cells were washed in
ice-cold
PBS,
collected
by
scraping,
pelleted
by
centrifugation
and
lysed
in
radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma; St Louis MO) as previously described (10). The lysates were then centrifuged at 16000×g for 10min at 4˚C. Supernatants were assayed for protein content using the Page 5
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bicinchoninic acid protein assay (Thermo Fisher Scientific). Equal amounts of lysate protein were diluted in gel loading buffer [375mM Tris-HCl, 6% sodium dodecyl sulfate (SDS), 48% glycerol, 9% β-mercaptoethanol and 0.03% bromophenol blue, pH 6.8], heated at 100°C for 5
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min and subjected to denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on pre-cast 4-20% Tris-glycine polyacrylamide gels using the Novex electrophoresis system (Life Technologies). Proteins were then electro-transferred to a
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polyvinylidene difluoride membrane (Millipore). For imumunodetection, membranes were first incubated in blocking buffer [5% nonfat milk in Tris-buffered saline containing 0.1% tween-20
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(TBST)] at room temperature for 1h and then with primary antibodies (Table 1) diluted in 5% nonfat milk/TBST overnight at 4˚C. The following day membranes were washed three times with TBST and incubated at room temperature for 1h with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit diluted in 5% nonfat milk/TBST. After three washes with TBST,
immunoreactive
proteins
were
detected
by
chemiluminescence
(HyGlo
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chemiluminescent reagent; Denville Scientific) and quantified by digital fluorescent densitometry (FluorChem E processor; ProteinSimple, San Jose , CA).
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mRNA abundance: Total RNA was isolated from cells using a commercial kit (Total RNA Isolation/NucleoSpin RNA II kit; Macherey-Nagel, Bethlehem, PA), treated with DNase (DNA-
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free; Life Technologies) and quantified by absorbance at 260nM. 300ng of total RNA was then reverse-transcribed with random hexamers using Superscript II Random Prime Synthesis kit for RT-PCR (Thermo Fisher Scientific, Grand Island, NY). Aliquots of cDNA were then used as template for quantitative reverse transcription polymerase chain reaction (qRT-PCR) with primers (Table 2) for transcripts of interest (designed using the PrimerExpress application; Applied Biosystems) on the StepOnePlus real-time PCR system (Applied Biosystems) using SYBR Green (Thermo Fisher Scientific) as the fluorescent detector to measure amplicon abundance in real-time. Abundance of specific mRNA relative to GAPDH mRNA was calculated Page 6
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using the ∆CT method [relative mRNA abundance = 2 − (CT gene of interest − CT GAPDH)]. Assays were validated for primer specificity and optimized to ensure the equivalent PCR efficiency for each
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primer pair.
P4/PR transcriptional activity via the progesterone response element: To assess P4/PR transcriptional activity, hTERT-HMA/B cells were transiently transfected with DNA containing the
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firefly luciferase (LUCFF) open reading frame controlled by a promoter comprised of tandem canonical progesterone response elements (PREs) (PRE2-LUCFF) and DNA containing the
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renilla LUC (LUCRN) controlled by the constitutively active cytomegalovirus (CMV) promoter (CMV-LUCRN). LUCFF and LUCRN activity were measured in cell lysates using the DualLuciferase reporter assay system (Promega, Madison WI) on a GloMax 20/20 luminometer (Promega).
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Statistical analyses: A minimum of n=3 replicates were used for each experimental condition and experiments were repeated in at least 3 time-separated studies. Normally distributed data (determined by Kolmogorov-Smirnov test) were compared using Student t test and analysis of
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variance (ANOVA) with post hoc Dunnett test. Non-normally distributed data were compared by Wilcoxon rank-sum test or Mann Whitney U test. Differences were considered statistically
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significant when the P value < 0.05.
RESULTS
Anti-inflammatory effects of P4/PR and forskolin: hTERT-HMA/B cells were maintained in the basal state (negligible PR levels and no PRE2-LUCFF response to P4) or induced to express equivalent amounts of PR-A and PR-B and then treated with P4 (100nM; Sigma) or vehicle [dimethyl sulfoxide (DMSO)] for 24h. Media was then changed and the cells were exposed to IL1ß (1ng/mL; Cell Signaling) in the presence and absence of P4 (replenished) and/or forskolin Page 7
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(10 µM; Sigma) for a further 24h. Total RNA and whole cell lysate were then prepared and used, respectively, to measure relative abundance of IL-8 mRNA by qRT-PCR, and protein by immunoblotting. As expected, IL-1ß increased the amount of IL-8 protein (Figure 1A) and mRNA
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(Figure 1B) in hTERT-HMA/B cells. This effect was inhibited by P4 only in cells induced to express PRs. Forskolin also inhibited IL-1ß-induced IL8 expression. The combination of P4 and forskolin produced a greater inhibition of IL-1ß-induced IL8 expression than either agent alone
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(Figures 1A and 1B). Inhibition of IL-1ß-induced IL8 expression by forskolin was dosedependent, mimicked by 8-bromoadenosine-cAMP (8-Br-cAMP; Sigma), a cAMP mimetic that is
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resistant to phosphodiesterase degradation (Figure 1C), and inhibited by H89 (10µM; Sigma), a PKA inhibitor, suggesting that anti-inflammatory actions of forskolin were mediated by activation of PKA by cAMP (Figure 1D).
Effect of forskolin and P4/PR on expression of labor-associated genes: The effect of
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forskolin on basal and P4- and IL-1ß-induced expression of genes encoding labor-associated inflammatory mediators and contraction-associated proteins (CAPs) was examined (Figure 2). For this experiment, hTERT-HMA/B cells were conditioned to express PR-A and PR-B (1:1) and
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exposed to P4 (100nM) or vehicle for 24h. The cells were then exposed to IL-1ß (1 ng/mL) in the presence of vehicle or increasing concentrations of forskolin (0.1-100 µM). Total RNA was
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then isolated and subjected to qRT-PCR to measure the extent of expression (assessed by the relative amount of cognate mRNAs) of genes encoding the oxytocin receptor (OXTR), the PGF2α receptor (PTGFR), connexin-43 (GJA1), IL-8 (IL8), prostaglandin-endoperoxide synthase-2 (PTGS2) and IL-6 (IL6) (Figure 2). Forskolin and P4/PR suppressed OXTR, GJA1, IL8, PTGS2 and IL6 expression in a forskolin concentration-dependent manner. P4/PR and forskolin had synergistic effects on the inhibition of IL-1ß-induced expression of IL8, PTGS2 and IL6 expression. There was no apparent synergistic effect of P4/PR and forskolin on OXTR and GJA1 expression, and neither agent affected expression of PTGFR. Page 8
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Effect of forskolin on P4/PR transcriptional activity: To examine the mechanism for synergy between P4/PR and cAMP in myometrial cells, the effect of forskolin on PR transcriptional
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activity was measured. To do this, hTERT-HMA/B cells were transiently co-transfected with PRE2-LUCFF and CMV-LUCRN as previously described (10). Cells were induced to express only PR-A, only PR-B, both PRs or left un-induced and exposed to P4 (100 nM) or vehicle for 24h.
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Media was then replenished with P4 or vehicle, and the cells exposed to forskolin (10 µM) for an additional 24h. LUCFF and LUCRN activity was then measured in whole cell lysates. As expected,
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P4 increased PRE2-LUCFF expression in cells induced to express PR-B but not in cells expressing only PR-A and cells lacking PRs (Figure 3A). Typical of PR-A trans-repressive activity, PR-A inhibited P4-induced PRE2-LUCFF expression mediated by PR-B. Forskolin did not alter P4/PR-induced expression of PRE2-LUCFF or PR-A trans-repression of PR-B at the PRE2LUCFF promoter.
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The effect of forskolin on P4/PR transcriptional activity was also examined using an endogenous P4/PR-responsive gene, FKBP5, whose promoter contains multiple PREs. We previously found that in hTERT-HMA/B cells treatment with P4 via PR-B but not PR-A induces
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FKBP5 expression (26). Expression of FKBP5 was not affected by forskolin (Figure 3B). Interestingly, unlike PRE2-LUCFF, PR-A did not trans-repress the activity of P4/PR-B-induced
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FKBP5 expression. These data are consistent with our previous studies showing that transrepressive activity of PR-A at the FKBP5 promoter is absent under basal conditions in hTERTHMA/B cells (26).
Effect of forskolin on pSer344/345-PRA generation: The hypothesis that cAMP increases myometrial cell P4/PR anti-inflammatory activity by inhibiting the generation of pSer344/345-PRA was tested. Cells were induced to express PR-A and PR-B and exposed to P4 or vehicle for 24h. Media and test agents were then replenished and some cultures were exposed to IL-1ß (1 Page 9
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ng/mL) and/or forskolin (10 µM) for a further 24h. Whole cell lysate was then prepared and used to determine the abundance of PR-A/B (total PRs), pSer344/345-PRs, IL-8 and GAPDH by immunoblotting (Figure 4). As expected, P4 increased pSer344/345-PRA and this was augmented
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by IL-1ß. Forskolin inhibited P4 and IL-1ß induced generation of pSer344/345-PRA.
Role of MAPKs in pSer344/345-PRA generation: The mechanism underlying the generation of pSer344/345-PRA in response to P4 and IL-1ß and its inhibition by forskolin was further explored
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by determining the role of MAPKs in catalyzing the generation of pSer344/345-PRA in hTERT-
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HMA/B cells. Generation of pSer344/345-PRA was abolished by the pan-RAF-kinase inhibitor LY3009120 (10 µM) that inhibits the activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p38 kinase, and stress-activated protein kinase/c-Jun N-terminal Kinase (SAPK/JNK), suggesting that these MAPKs catalyze pSer344/345-PRA formation (Figure 5A). The effect of forskolin on ERK1/2, p38 and SAPK/JNK content and activation (indicated by
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the level of respective phospho-forms) in hTERT-HMA/B cells treated to express PR-A and PR-B and then exposed to P4 (100nM), IL-1ß (1ng/mL) and various concentrations of forskolin was determined. Forskolin inhibited P4- and P4/IL-1ß-induced pSer344/345-PRA in a concentration-
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dependent manner (Figure 5B). The decrease in pSer344/345-PRA was associated with a parallel decrease in pThr202/Tyr204-ERK1/2 and pThr183/Tyr185-SAPK/JNK but not pThr180/Tyr182-p38.
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Further analyses were performed using U0126 (20µM) and SP600125 (100µM), small molecule inhibitors of ERK1/2 and SAPK/JNK, respectively. For these experiments, hTERT-HMA/B cells were exposed to U0126, SP600125 or vehicle for 1h and then to P4 for a further 1h. The 2h (vs 24h) time of exposure was chosen to avoid toxic effects of long-term (24h) exposure to U0126 and SP600125. Generation of pSer344/345-PRA and -PRB was inhibited by the SAPK/JNK inhibitor, SP600125, but not by the ERK1/2 inhibitor, U0126 that specifically decreased the level of phospho-ERK1/2 (Figure 5C). We previously showed that P4 induces the generation of pSer344/345-PRA and -PRB during the first 1-2h of exposure to P4 (9). Page 10
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Mechanism for cAMP inhibition of MAPK activity: Studies in other cell types suggest that cAMP inhibits MAPK activation by inducing the expression of DUSP1 that encodes dual specific
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protease-1 (DUSP1), a phosphatase that inactivates (by de-phosphorylation) MAPKs (27-30). It is also possible that anti-inflammatory actions of P4/PR are mediated by increased DUSP1 expression. We therefore determined whether P4/PR and forskolin affect DUSP1 expression
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(based on DUSP1 mRNA levels measured by qRT-PCR) in hTERT-HMA/B cells treated as described in Figure 2. P4/PR and forskolin increased DUSP1 expression. P4/PR caused a leftand up-shift in the forskolin concentration-response (Figure 6A).
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To determine whether DUSP1 mediates the inhibitory effect of forskolin on pSer344/345-PRA generation, its levels were decreased by RNA interference (RNAi) in hTERT-HMA/B cells by transfecting the cells with short interfering RNAs (Stealth siRNAs; Stealth ID:VHS40581 and VHS40583; Thermo Fisher) targeting the open reading frame of DUSP1 or with scrambled
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siRNAs (negative control). The cells were then induced to express PR-A and PR-B and exposed to P4 (100 nM), IL-1ß (1 ng/mL) and forskolin (10 µM) for 24h. Whole cell lysate was then prepared and subjected to immunoblotting to determine the levels of pSer344/345-PRA and
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pSer359-DUSP1 (Figure 6B). DUSP1 knock down by RNAi decreased pSer359-DUSP1. The decrease in DUSP1 was associated with increased pSer344/345-PRA in P4 treated cells and
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attenuated the inhibitory effects of forskolin on pSer344/345-PRA.
DISCUSSION
It is well-established that the cAMP/PKA signaling cascade promotes myometrial cell relaxation by decreasing intracellular free Ca2+ and preventing phosphorylation/activation of the myosin light chain (31-33). Consequently, this signaling pathway is thought to play a central role in promoting relaxation of the myometrium necessary for pregnancy. Indeed, various hormones acting via Gαs-linked receptor that activate adenylyal cyclase, have been implicated as
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contributing, via cAMP, to myometrial quiescence during pregnancy. However, cAMP signaling alone is not sufficient to maintain myometrial relaxation because it cannot compensate for the onset of labor triggered by P4/PR withdrawal. Moreover, the management of preterm labor with
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ß2-agonists has severe potentially life-threatening side effects on the maternal cardiovascular system and has limited tocolytic effectiveness due to pharmacologic down-regulation of adrenergic receptors in myometrial cells. Thus, although cAMP signaling promotes myometrial
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cell relaxation, this pathway is not sufficient to block labor in the context of P4 withdrawal. Instead, several studies suggest that cAMP signaling in myometrial cells functions in concert
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with P4/PR signaling to promote uterine quiescence. Chen et al.(8) reported that cAMP (induced by forskolin) increased the anti-inflammatory actions of P4 [likely acting via the glucocorticoid receptor (GR) in their model system] in primary cultures of term myometrial cells, and studies in breast cancer cells show that cAMP qualitatively and qualitatively modulates PR transcriptional activity (23,34). This evidence supports a functional cross-talk between cAMP and P4/PR
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signaling in myometrial cells that is distinct from effects of cAMP/PKA on the contractile apparatus and may be critical for the maintenance of pregnancy by modulating PR transcriptional activity. The present studies tested this hypothesis by focusing on the effect of
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cAMP (induced by forskolin) on the capacity for P4/PR to exert anti-inflammatory actions in myometrial cells and how its effects may be mediated by PR phosphorylation. Studies were
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performed in a human myometrial cell line, hTERT-HMA/B, genetically modified to allow experimental manipulation of PR-A and PR-B levels. IL-1ß was used as a prototypical pro-labor inflammatory stimulus, and expression of IL8 was measured as a prototypical readout for IL-1ßinduced pro-inflammatory/pro-labor action. IL-8 is a potent chemokine that promotes tissue-level inflammation and IL8 expression in myometrial cells increases in association with the onset of labor (34-36). cAMP signaling was induced by exposing cells to forskolin, an activator of adenylyl cyclase that increases intracellular levels of cAMP.
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We found that P4/PR and forskolin inhibited IL-1ß-induced IL8 expression via independent, yet functionally linked and synergistic, pathways (Figure 1). The combination of P4/PR and cAMP signaling promoted a gene expression profile consistent with myometrial relaxation and
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uterine quiescence based on decrease in expression of OXTR, GJA1, PTGS2, IL8 and IL6 (Figure 2). The data support the hypothesis that cAMP and P4/PR signaling in myometrial cells synergize to inhibit the response of myometrial cells to pro-labor/pro-inflammatory stimuli and
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affect expression of specific genes to promote uterine quiescence. This effect, coupled with cAMP-induced PKA-mediated inhibition of myometrial cell contraction may be critical for
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maintaining uterine quiescence during pregnancy.
We examined the mechanism by which forskolin increased the capacity for P4/PR to inhibit responsiveness to IL-1ß. In this regard, our studies extended those of Chen et al. (8) who proposed that, in primary myometrial cell cultures forskolin increased the capacity for P4 to inhibit IL-1ß-induced PTGS2 expression by increasing PR transcriptional activity. They found
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that forskolin increased the capacity for P4 to induced expression of FKBP5 and 11ßHSD1 expression, and PRE-LUC expression in cells transfected to over-express PR-B. The FKBP5 promoter contains multiple canonical PRE/glucocorticoid receptor response elements and its
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expression is induced by PR and GR. In this context, it is notable that P4 actions reported by Chen et al. were likely mediated via the GR, especially as the concentration of P4 used to exert
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an anti-inflammatory effects and drive gene expression was 100 µM, which is high (we used 100 nM) and likely sufficient to activate the GR. We therefore, re-examined the capacity for forskolin to affect P4/PR transcriptional activity using our hTERT-HMA/B cell model in which levels of PRA and PR-B were experimentally controlled. We found that, as expected, P4 induced PRE2LUCFF and FKBP5 expression and that this occurred only in cells induced to express PR-B, and that P4/PR-A trans-repressed P4/PRB-induced PRE2-LUCFF expression. Importantly, forskolin did not affect the capacity for P4/PR-B to induce expression of PRE2-LUCFF and FKBP5 or the capacity for PR-A to trans-repress PR-B activity at the PRE2-LUCFF promoter (Figure 3). Thus, Page 13
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our data suggest that effects of forskolin on P4/PR anti-inflammatory activity were not mediated by changes in canonical PR/PRE transcriptional activity. Recent studies suggest that P4/PR transcriptional activity in myometrial cells can occur by a
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non-canonical pathway whereby the PRs interact directly with other transcription factors (37-39), and that this is controlled via site-specific serine phosphorylation (37). Importantly, we previously found that pSer344/345-PRA inhibits net P4/PR anti-inflammatory activity in hTERT-
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HMA/B cells and that levels of pSer344/345-PRA were higher in laboring compared to quiescent term myometrium (9). Importantly, we found that in explant cultures of term myometrium and in
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hTERT-HMA/B cells, the generation of pSer344/345-PRA was P4-dependent and induced by IL-1ß. Those data led to the hypothesis that human parturition involves the induction of protein kinase activity in myometrial cells in response to pro-inflammatory stimuli that catalyzes pSer344/345-PRA generation, and that pSer344/345-PRA causes functional withdrawal of P4/PR anti-inflammatory activity leading to tissue level inflammation. Interestingly, our data suggest that inhibition of
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pSer344/345-PRA generation in myometrial cells is a hallmark of uterine quiescence, and in this context our finding that forskolin inhibited the generation of pSer344/345-PRA in hTERT-HMA/B cells further supports the notion that cAMP signaling promotes the quiescent phenotype in
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myometrial cells. Our finding that forskolin inhibited expression of OXTR, GJA1, IL8, IL6 and PTGS2 expression further supports this hypothesis and that synergism between the P4/PR and
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cAMP/PKA pathways extends beyond anti-inflammatory actions to affect a specific cassette of contraction-related and inflammatory genes. We found that the generation of pSer344/345-PRA in hTERT-HMA/B cells was induced by the SAPK/JNK kinase. This is consistent with the hypothesis that human parturition involves inflammation-induced functional P4/PR withdrawal mediated by the generation of pSer344/345PRA in response to stress-associated pro-inflammatory stimuli. Our finding that forskolin inhibited pSer344/345-PRA generation suggests that cAMP decreases SAPK/JNK activity in myometrial cells. One mechanism for this effect could be via cAMP-induced expression of Page 14
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DUSP1 (40). Previous studies have shown that P4 increases DUSP1 expression in myometrial cells, and that knockdown of DUSP1 abolished the capacity for P4 to inhibit IL-1ß-induced c-Jun activation and COX2 expression (38,41). Other studies showed that treatment of mice with
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phosphodiesterase inhibitors that elevate the level of cAMP, increased DUSP1 expression in different cell types, and that anti-inflammatory effects of phosphodiesterase inhibitors were significantly lower in DUSP1 knockout mice (28,42-44). We found that in hTERT-HMA/B cells P4/PR and forskolin increased DUSP1 expression in a cooperative manner, and that inhibition
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of DUSP1 by RNAi increased P4-induced pSer344/345-PRA and attenuated the inhibitory effects of forskolin on pSer344/345-PRA generation (Figure 6). These findings suggest that cAMP inhibits
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pSer344/345-PRA generation in myometrial cells, at least in part, by increasing DUSP1 expression and/or activity. Thus, consistent with the conclusions of Lei et al. (38), DUSP1 may be a key mediator of the cross-talk between cAMP and P4/PR signaling in myometrial cells and suppresses responsiveness to pro-inflammatory stimuli that function via MAPKs, and especially
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SAPK/JNK. This synergism may be critical for the maintenance of uterine quiescence for most of pregnancy. Further studies of this cross-talk may reveal novel therapeutic strategies to
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promote uterine quiescence and prevent preterm labor.
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Acknowledgments
We thank Lora J Mesiano for administra ive and editorial assistance. This work was funded by grants to SM by the March of Dimes Prematurity Center, Ohio Collaborative, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (HD069819).
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FIGURE 1
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FIGURE 2
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FIGURE 3
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FIGURE 4
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FIGURE 5
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FIGURE 6
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TABLE 1: Antibodies used for immunoblotting Manufacturer
Catalogue No.
Dilution
PR-A/B
Dako
M3568
1:1000
pSer344/345-PRA/B
Cell Signaling Technology
12783
1:1000
pSer359-DUSP1
Cell Signaling Technology
2857
1:1000
ERK1/2
Cell Signaling Technology
4696
1:1000
pThr202/Tyr204-ERK1/2
Cell Signaling Technology
9101
1:1000
p38
Cell Signaling Technology
9212
1:1000
pThr180/Tyr182-p38
Cell Signaling Technology
9211
1:1000
SAPK/JNK
Cell Signaling Technology
9252
1:1000
pThr183/Tyr185-SAPK/JNK
Cell Signaling Technology
9255
1:1000
Mouse IgG
Cell Signaling Technology
7076
1:3000
Rabbit IgG
Cell Signaling Technology
7074
1:3000
IL-8
R&D systems
MAB208
1:1000
SantaCruz Biotechnology
32233
1: 100000
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GAPDH
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Protein Target
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TABLE 2: Primer sequences for qRT-PCR Forward Primer
Reverse Primer
IL8
TGGCAGCCTTCCTGATTTCT
TTA GCA CTC CTT GGC AAA ACT G
PTGS2
ATGTTCCACCCGCAGTACAGA
CAGCATAAAGCGTTTGCGGTA
FKBP5
ATGCCATTTACTGTGCAAACCAG
AAGAGAGTTGCATTCGAGGGAA
GAPDH
TTGCCATCAATGACCCCTTCA
CGCCCCACTTGATTTTGGA
GJA1
TGG CCT TCT TGC TGA TCC A
TTT GCA AGT GTA AAC AGC ACT CAA
OXTR
CTG GAC GCC TTT CTT CTT CGT
GAA GGC CGA GGC TTC CTT
PTGFR
AGCAGTTTCAAAACTCTACCATGG
TGCCAATATTCTTTGCACCTATCA
IL6
TAGCCGCCCCACACAGA
CCGTCGAGGATGTACCGAAT
DUSP1
GCCACCATCTGCCTTGCTTA
TTCACAAACTCAAAGGCCTCG
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Gene
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FIGURE LEGENDS
Figure 1: Anti-inflammatory effects of P4/PR and forskolin. Effect of P4/PR, forskolin and 8Br-cAMP on IL-1ß-induced IL8 expression in hTERT-HMA/B cells. A: Immunoblot analyses of
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PR-A/B (total PRs), GAPDH and IL-8 in whole cell lysate from hTERT-HMA/B cells under basal conditions or induced to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) and forskolin (10 µM) for 24h. B: Relative
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abundance of IL8 mRNA measured by qRT-PCR in hTERT-HMA/B cells treated as described for A. C: Immunoblot analysis for PR-A/B (total PRs), GAPDH and IL-8 in whole cell lysate from
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hTERT-HMA/B cells treated to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) and varying concentrations of forskolin (upper) and 8-Br-cAMP (lower). D: Relative abundance of IL8 mRNA measured by qRT-PCR in hTERT-HMA/B cells treated to express PR-A (DOX: 100 ng/mL) and PR-B (DAH: 400 nM) and then exposed to P4 (100 nM) and/or IL-1ß (1 ng/mL) in the presence of H89 (10 µM; Sigma).
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Immunoblot data are representative of 3 time-separated experiments. Different gels are separated by white space. mRNA data are mean ± SE of n=3 and representative of 3 time-
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separated experiments. * P<0.05.
Figure 2: Effect of forskolin and P4/PR on expression of labor-associated genes. Relative
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abundance of RNAs (assayed by qRT-PCR) encoding OXTR, GJA1, PTGFR, IL-8, PTGS2, and IL-6 in total RNA from hTERT-HMA/B cells induced to express PR-A (DOX: 100 ng/mL) and PRB (DAH: 400 nM) and then exposed to P4 or vehicle for 24h and then exposed to IL-1ß (1 ng/ml) and increasing concentrations of forskolin (0-100 µM) for a further 24h. Mean ± SE; n=3.
Figure 3: Effect of forskolin on P4/PR transcriptional activity. A: Ratio of firefly LUC activity (from the PRE-LUC reporter) to renilla LUC activity (from CMV-LUC reporter) in hTERT-HMA/B
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cells under basal conditions or induced to express PR-A and/or PR-B, transiently co-transfected with PRE-LUC and CMV-LUC DNA and treated with vehicle or DOX (100 ng/mL) and/or DAH (400 nM) for 24h to induce PR-A and/or PR-B expression. Cells were then exposed to forskolin
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(10µM) and/or P4 (100nM) or vehicle for a further 24h. Cell lysate was then assayed for firefly and renilla luciferase activity. Mean ± SE n=3. B: Mean ± SE (n=3) relative abundance (measured by qRT-PCR) of mRNA encoding FKBP5 in hTERT-HMA/B cells treated as described
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to express PR-A and/or PR-B and then treated with P4 and/or forskolin. Mean ± SE (n=3).
Figure 4: Effect of forskolin on pSer344/345-PRA generation. Immunoblot analysis of PR-A/B
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(total PRs), pSer344/345-PR and IL-8 in hTERT-HMA/B cells conditioned to express PR-A and PR-B and then treated with vehicle or P4 (100 nM). Cells were then exposed to IL-1ß (1 ng/ml) and/or forskolin (10 µM) (with P4 replacement) for additional 24h. Representative of 3 identical time-
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separated experiments.
Figure 5: Role of MAPKs in pSer344/345-PRA generation: hTERT-HMA/B cells were induced to express PR-A and PR-B and exposed to test agents to determine the role of MAPKs in
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pSer344/345-PRA generation. A: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and GAPDH in lysate from cells exposed to RAF-kinase inhibitor, LY30019120 (10 µM; inhibits the
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activation of all MAPKs) for 1h and then exposed to P4 (100 nM) and IL-1ß (1 ng/mL) for additional 24h. B: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and total- and phospho-MAPKs: ERK1/2; p38 and SAPK/JNK in lysate from cells exposed to P4 (100 nM) for 24h and then challenged with IL-1ß (1 ng/mL) and increasing concentrations of forskolin (0.1 100µM) for additional 24h. C: Left: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR, phospho-ERK1/2 and GAPDH in lysate from cells treated with U0126 (20 µM: inhibits generation of active phospho-ERK1/2 by inhibiting MEK1/2 kinase) and SP600125 (100 µM: inhibits SAPK/JNK activity) for 1h and then exposed to vehicle or P4 (100nM) for a further 1h. Page 25
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Right: Immunoblot analysis of PR-A/B (total PRs), pSer344/345-PR and GAPDH in lysate from cells treated with vehicle or increasing concentrations of SP600125 (25-100 µM) for 1h and then exposed to vehicle or P4 (100nM) for a further 1h. Different gels are separated by white space.
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Data are representative of 3 identical time-separated experiments.
Figure 6: Mechanism for forskolin inhibition of MAPK activity. A: Relative abundance of
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DUSP1 mRNA measured by qRT-PCR in hTERT-HMA/B treated with P4 (100 nM) and increasing concentrations of forskolin (0.1 - 100µM). Mean ± SE; n=3. B: Immunoblot analysis of
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PR-A/B (total PRs), pSer344/345-PR, GAPDH and phospho-DUSP1 in lysate from cells transiently transfected with siRNAs targeting the DUSP1 open reading frame or siRNAs with scrambled sequence and then treated with P4 (100 nM) and forskolin (10 µM) for 24h. Representative of
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n=3 time-separated experiments.
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Progesterone (P4) and cAMP synergistically inhibit myometrial cells response to IL-1ß
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cAMP and P4/PR inhibit expression genes encoding contraction-associated
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factors. cAMP inhibits the phosphorylation of PR-A and induces the expression of DUSP1
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DUSP1 inhibits phosphorylation of PR-A
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