Progesterone and cAMP synergize to inhibit responsiveness of myometrial cells to pro-inflammatory/pro-labor stimuli
Introduction
Parturition (the process of birth) involves transition of the uterus from a quiescent to laboring state wherein the myometrium (the smooth muscle of the uterine wall) produces phasic and forceful contractions to become the engine for birth. In women, a major trigger for parturition is intrauterine inflammation and the onset of labor is associated with uterine tissue-level inflammation, involving the infiltration and activation of immune cells in the decidua, myometrium and cervix coupled with the local production of pro-inflammatory cytokines, especially interleukin (IL)-1β (IL-1β) and prostaglandins (PGs) that alter uterine cell—especially myometrial cell—phenotype to induce the labor (Goldenberg et al., 2008; Keelan et al., 2003; Osman et al., 2003; Romero et al., 2007; Thomson et al., 1999). Understanding the mechanisms controlling the responsiveness of myometrial cells to pro-labor/pro-inflammatory stimuli is therefore important for unravelling the hormonal and molecular interactions that promote uterine quiescence for most of pregnancy and transform the myometrium to the laboring state at parturition.
In vitro studies suggest that for most of pregnancy the steroid hormone progesterone (P4), via its interaction with the nuclear P4 receptor (PR) isoforms, PR-A and PR-B, blocks labor, in part, by inhibiting responsiveness of myometrial cells to pro-labor/pro-inflammatory stimuli (i.e., an anti-inflammatory effect) (Amini et al., 2016; Chen et al., 2014; Hardy et al., 2006; Peters et al., 2017; Tan et al., 2012). In all viviparous species studies so far, physiologic withdrawal of P4, or pharmacologic disruption of P4/PR signaling, induce parturition (Young et al., 2010). In women parturition is thought to be triggered by the functional loss of P4/PR anti-inflammatory activity in myometrial cells via changes in PR transcriptional activity (Mesiano et al., 2002, 2011). Our previous studies suggest that phosphorylation of PR-A at serine residues −344 and −345 (pSer344/345-PRA; numbers relative to amino acid 1 in PR-B) causes functional P4/PR withdrawal in myometrial cells (Amini et al., 2016). We found that the level of pSer344/345-PRA in term myometrium increased in association with active labor and that generation of pSer344/345-PRA in myometrial cells was P4-dependent and induced by IL-1β. 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 promote uterine quiescence by maintaining P4/PR anti-inflammatory activity. In this context, understanding the signaling cascade(s) responsible for the labor-associated generation of pSer344/345-PRA in myometrial cells is important for unraveling the physiologic control of human parturition.
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 (Chanrachakul et al., 2004; Price and Bernal, 2001). cAMP is produced by the adenylyl cyclase enzyme which is activated by hormones that bind to Gαs-coupled transmembrane receptors. In 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 responses (Skalhegg and Tasken, 2000). In smooth muscle cells PKA suppresses contractility by decreasing the intracellular concentration of free Ca2+ and inhibiting myosin light chain kinase activity (Billington et al., 2013; Morgado et al., 2012; Sanborn, 2007; Sanborn et al., 2005; Word, 1995; Yulia et al., 2016). cAMP may also contribute to the maintenance of pregnancy by exerting anti-inflammatory activity in myometrial cells, and by boosting P4/PR transcriptional activity (Chen et al., 2014). Studies in airway smooth 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-κB) and mitogen-activate protein kinases (MAPKs) (Oldenburger et al., 2012). In breast cancer cell lines cAMP affects P4/PR signaling in a quantitative and qualitative manner (Beck et al., 1992; Denner et al., 1990), and in myometrial cells derived from term uterus, forskolin, a bicyclic diterpene that increases intracellular cAMP by stimulating adenylyl cyclase, increases the anti-inflammatory activity of P4 (Chen et al., 2014). Thus, the functional interaction between P4/PR and cAMP signaling in myometrial cells may be critical for the maintenance of uterine quiescence during pregnancy by promoting myometrial cell relaxation and refractoriness to pro-inflammatory/pro-labor stimuli.
This study examined the functional interaction between P4/PR and cAMP signaling on the response of myometrial cells to pro-inflammatory/pro-labor stimuli using an immortalized human myometrial cell line, hTERT-HMA/B, that is genetically modified to allow experimental control of PR isoform levels (Tan et al., 2012) and in explant cultures of term myometrium. The hypothesis that synergism between P4/PR anti-inflammatory activity and cAMP signaling is mediated through their effect on MAPK signaling and control of PR phosphorylation, specifically pSer344/345-PRA generation, was tested. We found that cAMP signaling induced by forskolin increased P4/PR anti-inflammatory activity, in part, by inhibiting the generation of pSer344/345-PRA via increased expression of DUSP1, that encodes dual specificity protein phosphatase 1 (DUSP1), a phosphatase that de-phosphorylates, and therefore inactivates, MAPKs. In addition, we found that the generation of pSer344/345-PRA in hTERT-HMA/B cells was catalyzed by stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in response to IL-1β. Taken together the data suggest that cAMP and P4/PR signaling pathways synergistically inhibit the response of myometrial cells to pro-inflammatory stimuli, and that this is in part mediated by the inhibition of specific MAPKs (especially SAPK/JNK) by DUSP1. This may be a key mechanism for the maintenance of myometrial relaxation and uterine quiescence for most of pregnancy that could be exploited therapeutically to prevent preterm labor and its progression to preterm birth.
Section snippets
Methods
Cell culture: Studies were performed using the hTERT-HMA/B telomerase-immortalized human myometrial cell line 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 (Thermo Fisher Scientific, Waltham, MA). hTERT-HMA/B is a sub-clone of the hTERT-HM (Condon et al., 2002) that was genetically modified to
Results
Anti-inflammatory effects of P4/PR and forskolin: IL-1β increased the amount of IL-8 protein (Fig. 1A) and mRNA (Fig. 1B) in hTERT-HMA/B cells. This effect was inhibited by P4 only in cells induced to express PRs. Forskolin also inhibited IL-1β-induced IL8 expression. P4 and forskolin together produced greater inhibition of IL-1β-induced IL8 expression than either agent alone (Fig. 1A and B). Inhibition of IL-1β-induced IL8 expression by forskolin was dose-dependent, mimicked by
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 (Glorian and Limon, 2013; Lai et al., 2016; Lopez Bernal et al., 1995). 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,
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 Research Center Ohio Collaborative.
Disclosure statement
The authors have nothing to disclose.
Acknowledgements
We thank Lora J Mesiano for administrative and editorial assistance. This work was funded by grants to SM by the March of Dimes Prematurity Research Center Ohio Collaborative, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD069819).
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