Human Myometrial Cells Research Articles

Regulation of GTP-binding Protein (Gαs) Expression in Human Myometrial Cells

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.

Biphasic Erk1/2 activation sequentially involving Gs and Gi signaling is required in beta3-adrenergic receptor-induced primary smooth muscle cell proliferation

The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3min, the second at 8h). Erk1/2 activation at 3min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways.

Effect of corticotrophin-releasing hormone on connexin-43 phosphorylation and gap junction intercellular communication in human myometrial smooth muscle cells

To determine the effect of human corticotrophin-releasing hormone (CRH) on the expression of connexin-43 phosphate (P-Cx43) in human myometrial smooth muscle cells (SMCs) and the function of cell gap junction intercellular communication in SMCs. Human non-conceive myometial SMCs were cultured with different concentrations of CRH (0, 5.85, 58.5, 585 and 5850 pmol/L). Western blot was used to test P-Cx43 and Cx43 non-phosphate (NP-Cx43) of protein expression. Cell scratch was used to test cell gap junction intercellular communication opening status in human myometrial SMCs. Compared with the control group, the expression of P-Cx43 was higher in the CRH groups (P<0.01), and was concentration-dependent. There was no significant difference in NPCx43 between the control group and the CRH groups (P>0.05). The transmission of cell layers in the CRH groups was higher than that in the control group (P<0.01), and as the concentration of CRH increased, the time was concentration-dependent (P<0.01). CRH can enhance the expression of P-Cx43 and the function of gap junction intercellular communication in the primary cultured myometrial SMCs.

Development and validation of primary human myometrial cell culture models to study pregnancy and labour

BackgroundThe development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated.MethodsMyometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed.ResultsWe demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10.ConclusionsIsolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

TGF beta family members function in uterine healthy and fibrotic smooth muscle cells

Uterine leiomyomas are the most common benign tumors of fertile women and the most common indication for hysterectomy. Despite the high prevalence, significant health problems, and huge economical impact on the healthcare system, relatively little is understood about the etiology and pathophysiology of uterine leiomyoma (1). Consequently, medical treatments are still limited (2). The role of the growth factors as ultimate mediators of the steroids hormone is evident in the modulation of the cell proliferation and the morphological cells appearance (3). Activin-A and myostatin are growth factors belonging to TGF-β super family expressed and acting in myometrial (4,5) and leiomyoma cells (6) We aimed to explore the functions of activin and myostatin in human myometrial and leiomyoma cells. First we tested both Smad and non-Smad signaling pathways by western blot. We found that activin-A and myostatin can activate only Smad signaling pathway in both myometrial and leiomyoma cells. Next we explored the effect on cell proliferation and on fibrotic phenotype. We found that activin-A and myostatin are able to suppress primary myometrial cell proliferation but they cannot alter the proliferation of leiomyoma cells. In the next step, we found that activin-A can significantly increase fibronectin expression in leiomyoma cells. Those above results suggest that activin-A and myostatin may express antiproliferative and/or fibrotic effects depending on the cell types by activating Smad signaling pathway.

475: Vitamin D reverses oral bacteria induced increases in inflammatory markers and contractile associated factors in human myometrial cells co-cultured with monocytes

475: Vitamin D reverses oral bacteria induced increases in inflammatory markers and contractile associated factors in human myometrial cells co-cultured with monocytes

Expression of the GTP-Binding Protein Gαs in Human Myometrial Cells is Regulated by Ubiquitination and Protein Degradation: Involvement of Proteasomal Inhibition by Trichostatin A

In this study, we show that myometrial transcriptional complexes consisting of Sp1, Sp3, histone deacetylase (HDAC)1/2, RbAp48, and mSin3A are recruited to 4 out of the 6 Sp1-4 sites within the Gαs promoter. Moreover disruption in the binding of these complexes via mithramycin administration results in a substantial decrease in expression of Gαs proteins in myometrial cell cultures. In many instances, these transcriptional regulatory complexes repress expression of genes having a high CG content within their promoter region. This repression can be attenuated by inhibition of HDAC activity by the class I/II HDAC inhibitor trichostatin A (TSA) resulting in increased gene transcription. However, although a substantial increase in Gαs protein levels was observed upon administration of TSA to primary cultures of human myometrial cells, this was not preceded by an increase in messenger RNA (mRNA) and thus an elevation in gene transcription. Importantly the increase in Gαs protein levels occurred via ubiquitination and inhibition of proteasomal activity, indicating that this pathway is also involved in regulating Gαs protein expression during pregnancy and parturition.

Progesterone Acts via the Nuclear Glucocorticoid Receptor to Suppress IL-1β-Induced COX-2 Expression in Human Term Myometrial Cells

Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via the inhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsible for this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1β) inhibits progesterone driven PRE activation via p65 activation and that IL-1β reduced progesterone driven gene expression (FKBP5). Conversely, we found that the activity of a p65-driven NFκB reporter construct was reduced by overexpression of progesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA and progesterone suppressed IL-1β-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-tagged PRB, but not PRA, bound to p65 and that in IL-1β-treated cells, with no overexpression of either PR or p65, activated p65 bound to PR. However, we found that the ability of MPA to repress IL-1β-driven COX-2 expression was not enhanced by overexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects of progesterone and MPA, the specific PR antagonist, Org31710, did not, suggesting that progesterone and MPA act via GR and not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repress IL-1β-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1β-driven COX-2 activation and that although the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression it does not seem to be responsible for progesterone repression of IL-1β-induced COX-2 expression.

Proliferative Action of the Androgen Receptor in Human Uterine Myometrial Cells—A Key Regulator for Myometrium Phenotype Programming

During pregnancy, the myometrium undergoes a phenotype programming starting from an early proliferative stage, to an intermediate synthetic stage, to a late contractile stage, after which the cells commit to labor. Steroid receptors play important roles in regulating myometrial cell phenotype during pregnancy, although detailed mechanisms are not fully defined. The aim of the study was to investigate the expression and function of the androgen receptor (AR) in myometrial cells during pregnancy. Human primary myometrial cells, immortalized myometrial cells, rat pregnant and tubal ligation models were used. Immunohistochemistry, Western blot and real-time PCR, cell proliferation, and flow cytometry assays were applied. The AR is highly expressed in the proliferative stage of pregnancy, starts to decrease in the synthetic stage, and reaches the lowest levels in the contractile stage. Both the mechanical stretch by the growing fetus and the decreased ratio of progestin:estrogen are responsible for AR protein reduction. AR regulates myometrial cell proliferation ligand-independently. Decreased AR expression delays the G(1)-S phase transition of human myometrial cell cycling and reduces expression of several cyclins. These AR actions are mediated through reducing IGF-I receptor protein stability, thus weakening PI3K/Akt signal cascade downstream of IGF-I. AR is required for IGF-I receptor protein stability by preventing the IGF-I receptor from ubiquitylation and protein degradation through both proteosomal and lysosomal pathways. AR is a key regulator for myometrial cell proliferation, suggesting its critical role in myometrium phenotype programming during pregnancy.

The microRNA (miR)-199a/214 Cluster Mediates Opposing Effects of Progesterone and Estrogen on Uterine Contractility during Pregnancy and Labor

Progesterone (P(4)) and estradiol-17β (E(2)) play critical and opposing roles in regulating myometrial quiescence and contractility during pregnancy and labor. Although these contrasting hormonal effects are likely mediated via differential regulation of inflammatory and contractile genes, the underlying mechanisms remain incompletely understood. Recently we discovered that targets of the microRNA (miR)-200 family, transcription factors zinc finger E-box binding homeobox (ZEB)-1 and ZEB2, serve as P(4)/progesterone receptor-mediated regulators of uterine quiescence during pregnancy. In the present study, we found that levels of the clustered miRNAs, miR-199a-3p and miR-214, were significantly decreased in laboring myometrium of pregnant mice and humans and in an inflammatory mouse model of preterm labor, whereas the miR-199a-3p/miR-214 target, cyclooxygenase-2, a critical enzyme in synthesis of proinflammatory prostaglandins, was coordinately increased. Overexpression of miR-199a-3p and miR-214 in cultured human myometrial cells inhibited cyclooxygenase-2 protein and blocked TNF-α-induced myometrial cell contractility, suggesting their physiological relevance. Notably, E(2) treatment of ovariectomized mice suppressed, whereas P(4) enhanced uterine miR-199a-3p/214 expression. Intriguingly, these opposing hormonal effects were mediated by ZEB1, which is induced by P(4), inhibited by E(2) and activates miR199a/214 transcription. Together, these findings identify miR-199a-3p/miR-214 as important regulators of myometrial contractility and provide new insight into strategies to prevent preterm birth.

Epigenome & uterine fibroids: distinct patterns of chromatin modifying genes in human myometrial stem cells from normal versus fibroid uteri

Uterine fibroids are a common indication for hysterectomy in women.An epigenetic chromatin modification in early life (in utero/neonatal sensitive period) leading to an adult onset of fibroids may be a potential mechanism for its pathogenesis. In this study we investigated the expression of genes involved in epigenetic chromatin modification in human myometrial stem cells from normal versus fibroid uteri as well as fibroid stem cells from fibroid tumor lesions.

Oxytocin-Stimulated NFAT Transcriptional Activation in Human Myometrial Cells

Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca2+ stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1–C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.

Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells.

Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE2 and PGF2α, matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM–100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca2+]i mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.

Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine

Ethnopharmacological relevancePregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation. Aim of studyObtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level. Materials and methodsSemi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model. ResultThe plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure. ConclusionThe folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors1

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.

Cyclic AMP increases COX‐2 expression via mitogen‐activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF1-2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.

PP042. Anti-hypertensive drugs hydralazine, clonidine and labetalol improve trophoblast integration into endothelial cellular networks in vitro

Preeclampsia is an exaggerated maternal inflammatory state with over-expression of placental soluble fms-like tyrosine kinase 1 (sFlt-1). It is also associated with shallow trophoblast invasion and inadequate transformation of uterine spiral arteries. Antihypertensive drugs administrated in preeclampsia to control blood pressure have been reported to regulate placental and circulating cytokine production from women with preeclampsia. Whether they could modulate the interaction between trophoblast and endothelial cells are not investigated. This study is to examine the effect of pharmacological dose of anti-hypertensive hydralazine, clonidine and labetalol on trophoblast cell integration into inflammatory TNF-a pre-exposed endothelial cellular networks. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose (0.5ng/ml) inflammatory TNF-a or TNF-a plus sFlt-1 (100ng/ml) for 24hours. These cells were labelled with red fluorescence and seeded on a 24-well culture plate coated with Matrigel. Endothelial tubular structures appeared within 4hours. Green fluorescent-labelled HTR-8/SVneo trophoblast cells were then co-cultured with endothelial cells, with (or without) hydralazine (10μg/ml), clonidine (1.0μg/ml) or labetalol (0.5μg/ml). Red and green fluorescent images were captured after 24hours. Drug effect on HTR-8 cells integration into endothelial cellular networks was quantified by Image Analysis software. The conditioned media were also collected to measure concentrations of free VEGF, PLGF and sFlt-1 by ELISA. When HTR-8/SVneo trophoblast cells were co-cultured with TNF-a pre-incubated endothelial cells, hydralazine and clonidine can significantly increase the trophoblast integration into endothelial cellular networks. This increase was not seen if co-cultured with normal endothelial cells (without TNF-a pre-incubation) or with TNF-a plus sFlt-1 treated endothelial cells. Labetalol could increase the HTR-8 cell integration into all endothelial cellular networks (with TNF-a and TNF-a plus sFlt-1 treatment). There was no effect of any drug on the trophoblast invasion in the absence of TNF-a. In the conditioned medium, sFlt-1 was down-regulated by hydralazine and clonidine but not by labetalol. A decrease in PLGF was seen in normal endothelial cell groups but not seen in pre-TNF-a or pre-TNF-a plus sFlt-1 endothelial cell groups. VEGF was not changed in all treatment groups. Some anti-hypertensive drugs may improve the cellular interaction between trophoblast and endothelial cells during pregnancy. An increase in sFlt-1 seen in human preeclampsia may deteriorate this effect. Hydralazine and clonidine can reduce the sFlt-1 concentration in the medium. Labetalol could increase trophoblast integration without decreasing sFlt-1, suggesting a mechanism independent of sFlt-1. Our in-vitrodata show that there is a potential effect of these agents on placental maternal arterial modelling. The drug effect on arterial/trophoblast interactions can be related to altered production of sFlt-1.

MicroRNA-200a serves a key role in the decline of progesterone receptor function leading to term and preterm labor

During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Previously, we discovered that miR-200 family members serve as progesterone (P(4))-modulated activators of contraction-associated genes in the pregnant uterus. In this study, we identified a unique role for miR-200a to enhance the local metabolism of P(4) in myometrium and, thus, decrease PR function during the progression toward labor. miR-200a exerts this action by direct repression of STAT5b, a transcriptional repressor of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. Notably, overexpression of miR-200a in cultured human myometrial cells (hTERT-HM) suppressed STAT5b and increased 20α-HSD mRNA levels. In uterine tissues of ovariectomized mice injected with P(4), miR-200 expression was significantly decreased, STAT5b expression was up-regulated, and 20α-HSD mRNA was decreased, but in 15 d postcoitum pregnant mice injected with the PR antagonist RU486, preterm labor was associated with increased miR-200a, decreased STAT5b, and enhanced 20α-HSD expression. Taken together, these findings implicate miR-200a as an important regulator of increased local P(4) metabolism in the pregnant uterus near term and provide insight into the importance of miR-200s in the decline in PR function leading to labor.

Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition

Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone.

Calreticulin has opposing effects on the migration of human trophoblast and myometrial endothelial cells

Calreticulin is a calcium binding, endoplasmic reticulum resident protein best known for its roles in intracellular calcium homeostasis and the quality control processes of the endoplasmic reticulum. There is evidence for a range of activities for calreticulin outside the endoplasmic reticulum, including in the cytosol, on the surface of different cells types and in the extracellular matrix. Recent evidence indicates that human pregnancy is a condition of elevated circulating calreticulin. Calreticulin was increased in the plasma of women throughout pregnancy compared to the non-pregnant state. Calreticulin was also further increased during the hypertensive disorder of human pregnancy, pre-eclampsia. To clarify the roles of circulating calreticulin in pregnancy and pre-eclampsia, the aim of this study was to determine the effects of exogenous calreticulin on two cell types that are relevant to normal human pregnancy and to pre-eclampsia. Human primary myometrial microvascular endothelial cells (UtMVEC-Myo) and the human trophoblast cell line, HTR8/SVneo, were cultured with exogenous calreticulin at concentrations (2 μg/ml and 5 μg/ml) comparable to that measured in maternal blood. The higher concentration of calreticulin significantly increased the migration of the UtMVEC-Myo cells, but significantly reduced the migration of the HTR8/SVneo cells. In the presence of only FGF, FBS and antibiotics calreticulin at 5 μg/ml significantly reduced the number of UtMVEC-Myo cells during in vitro culture for 120 h. These results demonstrate that exogenous calreticulin can alter both HTR8/SVneo and UtMVEC-Myo cell functions in vitro at a (patho-) physiologically relevant concentration. Increased calreticulin may also contribute to altered functions of both cell types during pre-eclampsia.