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The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (-12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (-38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PCF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process. [ABSTRACT FROM AUTHOR]

Progesterone (P 4 ), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P 4 /PR partially exerts these effects by tethering to NF-κB bound to their promot-ers, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P 4 /PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a corepressor that also interacts within the same chromatin complex as PR-B. In mouse myome-trium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in enrichment of repressive histone marks and increase in enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in expression of corresponding histone modifying enzymes. Myometrial tissues from P 4 -treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P 4 treatment of PR-expressing hTERT-HM cells enhanced CNOT1 expression and its recruitment to PR bound NF-κB-response elements within the CX43 and OXTR promoters. Furthermore, knockdown of CNOT1 significantly increased expression of contractile genes. These novel findings suggest that decreased expression and DNA-binding of the P 4 /PR-regulated transcriptional corepressor CNOT1 near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Inflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators., Methods: To determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry., Results: Upon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells., Conclusions: Our studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion. Video Abstract., (© 2023. The Author(s).)

Fetal development and parturition are precisely regulated processes that involve continuous crosstalk between the mother and the fetus. Our previous discovery that wild-type mice carrying steroid receptor coactivator (Src)-1 and Src-2 double-deficient fetuses exhibit impaired lung development and delayed labor, which indicates that the signals for parturition emanate from the fetus. In this study, we perform RNA sequencing and targeted metabolomics analyses of the lungs from fetal Src-1/-2 double-knockout mice and find that expression of arginase 1 (Arg1) is significantly decreased, accompanied by increased levels of the Arg1 substrate L-arginine. Knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. Moreover, treatment of human myometrial smooth muscle cells with L-arginine significantly inhibits spontaneous contractions by attenuating activation of NF-κB and downregulating expression of contraction-associated protein genes. Transcription factors GR and C/EBPβ increase transcription of Arg1 in an Src-1/Src-2-dependent manner. These findings provide new evidence that fetus-derived factors may play dual roles in coordinating fetal lung development and the initiation of labor., (© 2023 The Authors.)

Using cultured human trophoblast stem cells (hTSCs), mid-gestation human trophoblasts in primary culture, and gene-targeted mice, we tested the hypothesis that the multinucleated syncytiotrophoblast (SynT) serves a critical role in pregnancy maintenance through production of key immune modulators/checkpoint proteins (ICPs) under control of the O2-regulated transcription factor, NRF2/NFE2L2. These ICPs potentially act at the maternal-fetal interface to protect the hemiallogeneic fetus from rejection by the maternal immune system. Using cultured hTSCs, we observed that several ICPs involved in the induction and maintenance of immune tolerance were markedly upregulated during differentiation of cytotrophoblasts (CytTs) to SynT. These included HMOX1, kynurenine receptor, aryl hydrocarbon receptor, PD-L1, and GDF15. Intriguingly, NRF2, C/EBPβ, and PPARγ were markedly induced when CytTs fused to form SynT in a 20% O2 environment. Notably, when hTSCs were cultured in a hypoxic (2% O2) environment, SynT fusion and the differentiation-associated induction of NRF2, C/EBPβ, aromatase (CYP19A1; SynT differentiation marker), and ICPs were blocked. NRF2 knockdown also prevented induction of aromatase, C/EBPβ and the previously mentioned ICPs. Chromatin immunoprecipitation-quantitative PCR revealed that temporal induction of the ICPs in hTSCs and mid-gestation human trophoblasts cultured in 20% O2 was associated with increased binding of endogenous NRF2 to putative response elements within their promoters. Moreover, placentas of 12.5 days postcoitum mice with a global Nrf2 knockout manifested decreased mRNA expression of C/ebpβ, Pparγ, Hmox1, aryl hydrocarbon receptor, and Nqo1, another direct downstream target of Nrf2, compared with wild-type mice. Collectively, these compelling findings suggest that O2-regulated NRF2 serves as a key regulator of ICP expression during SynT differentiation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

After birth, the alveolar epithelium is exposed to environmental pathogens and high O 2 tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPβ and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPβ, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5 dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival., (© 2021 Federation of American Societies for Experimental Biology.)

Our previous research revealed that steroid receptor coactivators (Src)-1 and -2 serve a critical cooperative role in production of parturition signals, surfactant protein A and platelet-activating factor, by the developing mouse fetal lung (MFL). To identify the global landscape of genes in MFL affected by Src-1/-2 double-deficiency, we conducted RNA-seq analysis of lungs from 18.5 days post-coitum (dpc) Src-1 -/- /-2 -/- (dKO) vs. WT fetuses. One of the genes most highly downregulated (~4.8 fold) in Src-1/-2 dKO fetal lungs encodes 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyzes conversion of inactive 11-dehydrocorticosterone to the glucocorticoid receptor (GR) ligand, corticosterone. Glucocorticoids were reported to upregulate 11β-HSD1 expression in various cell types via induction of C/EBP transcription factors. We observed that C/ebpα and C/ebpβ mRNA and protein were markedly reduced in Src-1/-2 double-deficient (Src-1/-2 d/d ) fetal lungs, compared to WT. Moreover, glucocorticoid induction of 11β-hsd1, C/ebpα and C/ebpβ in cultured MFL epithelial cells was prevented by the SRC family inhibitor, SI-2. Cytokines also contribute to the induction of 11β-HSD1. Expression of IL-1β and TNFα, which dramatically increased toward term in lungs of WT fetuses, was markedly reduced in Src-1/-2 d/d fetal lungs. Our collective findings suggest that impaired lung development and surfactant synthesis in Src-1/-2 d/d fetuses are likely caused, in part, by decreased GR and cytokine induction of C/EBP and NF-κB transcription factors. This results in reduced 11β-HSD1 expression and glucocorticoid signaling within the fetal lung, causing a break in the glucocorticoid-induced positive feedforward loop., (© 2020 Federation of American Societies for Experimental Biology.)

The steroid hormones progesterone (P 4 ) and estradiol-17β (E 2 ), produced by the placenta in humans and the ovaries in rodents, serve crucial roles in the maintenance of pregnancy, and the initiation of parturition. Because of their critical importance for species survival, the mechanisms whereby P 4 and its nuclear receptor (PR) maintain myometrial quiescence during pregnancy, and for the decline in P 4 /PR and increase in E 2 /estrogen receptor (ER) function leading to parturition, are multifaceted, cooperative, and redundant. These actions of P 4 /PR include: (1) PR interaction with proinflammatory transcription factors, nuclear factor κB (NF-κB), and activating protein 1 (AP-1) bound to promoters of proinflammatory and contractile/contraction-associated protein (CAP) genes and recruitment of corepressors to inhibit NF-κB and AP-1 activation of gene expression; (2) upregulation of inhibitors of proinflammatory transcription factor activation (IκBα, MKP-1); (3) induction of transcriptional repressors of CAP genes (e.g., ZEB1). In rodents and most other mammals, circulating maternal P 4 levels remain elevated throughout most of pregnancy and decline precipitously near term. By contrast, in humans, circulating P 4 levels and myometrial PR levels remain elevated throughout pregnancy and into labor. However, even in rodents, wherein P 4 levels decline near term, P 4 levels remain higher than the K d for PR binding. Thus, parturition is initiated in all species by a series of molecular events that antagonize the P 4 /PR maintenance of uterine quiescence. These events include: direct interaction of inflammatory transcription factors (e.g., NF-κB, AP-1) with PR; increased expression of P 4 metabolizing enzymes; increased expression of truncated/inhibitory PR isoforms; altered expression of PR coactivators and corepressors. This article will review various mechanisms whereby P 4 acting through PR isoforms maintains myometrial quiescence during pregnancy as well as those that underlie the decline in PR function leading to labor. The roles of P 4 - and E 2 -regulated miRNAs in the regulation and integration of these mechanisms will also be considered., (Copyright © 2019 Mendelson, Gao and Montalbano.)

Defective placental implantation and vascularization with accompanying hypoxia contribute to preeclampsia (PE), a leading cause of maternal and neonatal morbidity and mortality. Genetic and epigenetic mechanisms underlying differentiation of proliferative cytotrophoblasts (CytTs) to multinucleated syncytiotrophoblast (SynT) are incompletely defined. The SynT performs key functions in nutrient and gas exchange, hormone production, and protection of the fetus from rejection by the maternal immune system. In this study, we used chromatin immunoprecipitation sequencing of midgestation human trophoblasts before CytT and after SynT differentiation in primary culture to analyze changes in binding of RNA polymerase II (Pol II) and of active and repressive histone marks during SynT differentiation. Our findings reveal that increased Pol II binding to promoters of a subset of genes during trophoblast differentiation was closely correlated with active histone marks. This gene set was enriched in those controlling immune response and immune modulation, including interferon-induced tetratricopeptide repeat and placenta-specific glycoprotein gene family members. By contrast, genes downregulated during SynT differentiation included proinflammatory transcription factors ERG1, cFOS, and cJUN, as well as members of the NR4A orphan nuclear receptor subfamily, NUR77, NURR1, and NOR1. Downregulation of proinflammatory transcription factors upon SynT differentiation was associated with decreased promoter enrichment of endogenous H3K27Ac and H3K9Ac and enhanced binding of H3K9me3 and histone deacetylase 1. However, promoter enrichment of H3K27me3 was low in both CytT and SynT and was not altered with changes in gene expression. These findings provide important insight into mechanisms underlying human trophoblast differentiation and may identify therapeutic targets for placental disorders, such as PE., (Copyright © 2019 Endocrine Society.)

The major surfactant protein, SP-A (a product of the SFTPA gene), serves as a marker of type II pneumocyte differentiation and surfactant synthesis. SFTPA expression in cultured human fetal lung (HFL) epithelial cells is upregulated by hormones that increase cyclic AMP (cAMP) and activate TTF-1/NKX2.1 and NF-κB. To further define mechanisms for type II cell differentiation and induction of SP-A, we investigated roles of microRNAs (miRNAs). Using microarray to identify differentially expressed miRNAs in HFL epithelial cells during type II cell differentiation in culture, we observed that members of the miRNA 199a (miR-199a)/miR-214 cluster were significantly downregulated during differentiation. Validated and predicted targets of miR-199a-3p/miR-199a-5p and miR-214, which serve roles in type II cell differentiation (COX-2, NF-κB p50/p65, and CREB1), and the CREB1 target, C/EBPβ, were coordinately upregulated. Accordingly, overexpression of miR-199a-5p, miR-199a-3p, or miR-214 mimics in cultured HFL epithelial cells decreased COX-2, NF-κB p50/p65, CREB1, and C/EBPβ proteins, with an associated inhibition of SP-A expression. Interestingly, overexpression of the EMT factor, ZEB1, which declines during cAMP-induced type II cell differentiation, increased pri-miR-199a and reduced the expression of the targets NF-κB/p50 and COX-2. Collectively, these findings suggest that the developmental decline in miR-199a/miR-214 in HFL causes increased expression of critical targets that enhance type II cell differentiation and SP-A expression., (Copyright © 2018 American Society for Microbiology.)

Dysregulation of human trophoblast invasion and differentiation with placental hypoxia can result in preeclampsia, a hypertensive disorder of pregnancy. Herein, we characterized the role and regulation of miR-1246, which is markedly induced during human syncytiotrophoblast differentiation. miR-1246 targets GSK3β and AXIN2, inhibitors of WNT/β-catenin signaling, which is crucial for placental development, and is predicted to target JARID2, which promotes silencing of developmentally regulated genes. Human cytotrophoblasts cultured in 20% O2 spontaneously differentiate to syncytiotrophoblast with induction of hCYP191A/aromatase, a marker of differentiation. miR-1246 was induced >150-fold during syncytiotrophoblast differentiation in 20% O2, whereas targets-GSK3β, AXIN2, and JARID2-were significantly decreased. However, when cytotrophoblasts were cultured in 2% O2, miR-1246 and aromatase induction were prevented. miR-1246 was significantly decreased in placentas of women with severe preeclampsia, whereas AXIN2, GSK3β, and JARID2 were increased, compared with normotensive subjects. To identify factors that regulate miR-1246, we investigated the redox-regulated transcription factor NRF2, which has predicted binding sites in the miR-1246 promoter. Intriguingly, NRF2 messenger RNA was upregulated during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in preeclamptic placentas. Moreover, NRF2 knockdown in cytotrophoblasts inhibited induction of miR-1246 and hCYP19A1, as well as transcription factors C/EBPβ and PPARγ, which are implicated in placental differentiation. Using chromatin immunoprecipitation-quantitative polymerase chain reaction, we found that binding of endogenous NRF2 to the miR-1246 and hCYP191A promoters increased during syncytiotrophoblast differentiation. Thus, NRF2 promotes syncytiotrophoblast differentiation by inducing C/EBPβ, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.

The mechanisms whereby progesterone (P 4 ), acting via the progesterone receptor (PR), inhibits proinflammatory/contractile gene expression during pregnancy are incompletely defined. Using immortalized human myometrial (hTERT-HM) cells stably expressing wild-type PR-A or PR-B (PR WT ), we found that P 4 significantly inhibited IL-1β induction of the NF-κB target genes, COX-2 and IL-8 P 4 -PR WT transrepression occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA polymerase II to COX-2 and IL-8 promoters. However, in cells stably expressing a PR-A or PR-B DNA-binding domain mutant (PR mDBD ), P 4 -mediated transrepression was significantly reduced, suggesting a critical role of the PR DBD. ChIP analysis of hTERT-HM cells stably expressing PR WT or PR mDBD revealed that P 4 treatment caused equivalent recruitment of PR WT and PR mDBD to COX-2 and IL-8 promoters, suggesting that PR inhibitory effects were not mediated by its direct DNA binding. Using immunoprecipitation, followed by MS, we identified a transcriptional repressor, GATA zinc finger domain-containing 2B (GATAD2B), that interacted strongly with PR WT but poorly with PR mDBD P 4 treatment of PR WT hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P 4 -PR WT transrepression of COX-2 and IL-8 Notably, GATAD2B expression was significantly decreased in pregnant mouse and human myometrium during labor. Our findings suggest that GATAD2B serves as an important mediator of P 4 -PR suppression of proinflammatory and contractile genes during pregnancy. Decreased GATAD2B expression near term may contribute to the decline in PR function, leading to labor., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Preterm birth remains the major cause of neonatal morbidity and mortality throughout the world. This is due, in part, to our incomplete understanding of the mechanisms that underlie the maintenance of pregnancy and the initiation of parturition at term. In this article, we review our current knowledge of the complex, interrelated and concerted mechanisms whereby progesterone maintains myometrial quiescence throughout most of pregnancy, as well as those that mediate the upregulation of the inflammatory response and decline in progesterone receptor function leading to parturition. Herein, we review findings that demonstrate a role of the fetus in the timing of birth. Specifically, we focus on our own studies indicating that maturation of the fetal lung and enhanced secretion of the surfactant components, surfactant protein A (SP-A) and the potent inflammatory glycerophospholipid, platelet-activating factor (PAF), initiate a signaling cascade culminating in parturition. Our studies suggest an essential role of steroid receptor coactivators, SRC-1 and SRC-2, which activate expression of genes encoding SP-A and LPCAT1. LPCAT1 is a key enzyme in the synthesis of PAF, as well as DPPC, a highly surface-active glycerophospholipid component of surfactant. Thus, we describe a novel pathway through which the fetus contributes to the initiation of labor by signaling the mother when its lungs have achieved sufficient maturity for survival in an aerobic environment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Dysregulation of human trophoblast invasion and differentiation can result in preeclampsia (PE), a hypertensive disorder of pregnancy with significant morbidity and mortality for mother and offspring. miRNA microarray analysis of RNA from human cytotrophoblasts (CytT), before and after differentiation to syncytiotrophoblast (SynT) in primary culture, revealed that members of miR-515 family-including miR-515-5p, miR-519e-5p, miR-519c-3p, and miR-518f, belonging to the primate- and placenta-specific chromosome 19 miRNA cluster ( C19MC )-were significantly down-regulated upon human SynT differentiation. The proto-oncogene, c-MYC, which declines during SynT differentiation, interacted with E-boxes upstream of pri-miR-515-1 and pri-miR-515-2 , encoding these mRNAs, to enhance their expression. Predicted targets of miR-515-5p, known to be critical for human SynT differentiation, including hCYP19A1/aromatase P450, glial cells missing 1 (GCM1), frizzled 5 (FZD5), WNT2, Sp1, and estrogen receptor-α (ERα) mRNA, were markedly up-regulated during SynT differentiation. Notably, overexpression of miR-515-5p in cultured primary human trophoblasts impaired SynT differentiation and specifically decreased expression of hCYP19A1, GCM1, and Fzd5, which were validated as its direct targets. Interestingly, miR-515-5p levels were significantly increased in PE placentas, whereas mRNA and protein levels of targets, hCYP19A1, GCM1, and FZD5, were significantly decreased, compared with placentas of normotensive women. Thus, miR-515-5p may serve a key role in human trophoblast differentiation; its aberrant up-regulation may contribute to the pathogenesis of PE., Competing Interests: The authors declare no conflict of interest.

Context: The initiation of term and preterm labor is associated with an up-regulated inflammatory response in myometrium; however, the underlying signaling pathways remain incompletely defined., Objective: To define the regulatory mechanisms that mediate the increased myometrial inflammatory response leading to labor, we investigated the roles of microRNAs (miRNA/miR)., Design and Setting: Human myometrial tissues, isolated smooth muscle cells, and animal models were used to study miR-181a regulation of uterine inflammatory pathways and contractility., Patients: Myometrial tissues from 15 term pregnant women undergoing elective cesarean section (not in labor) and 10 term pregnant women undergoing emergency cesarean section (in labor) were used., Results: Expression of the highly conserved microRNA, miR-181a, was significantly decreased in mouse and human myometrium during late gestation. By contrast, the putative miR-181a targets, TNF-α, and estrogen receptor (ER)-α, and the validated target, c-Fos, key factors in the inflammatory response leading to parturition, were coordinately up-regulated. In studies using human myometrial cells, overexpression of miR-181a mimics repressed basal as well as IL-1β-induced TNF-α, C-C motif chemokine ligand 2 and 8 expression, whereas the expression of the antiinflammatory cytokine, IL-10, was increased. Overexpression of miR-181a dramatically inhibited both spontaneous and IL-1β-induced contraction of human myometrial cells. Notably, miR-181a directly targeted ERα and decreased its expression, whereas estradiol-17β reciprocally inhibited expression of mature miR-181a in myometrial cells., Conclusions: Thus, increased estradiol-17β/ERα signaling in myometrium near term inhibits miR-181a, resulting in a further increase in ERα and proinflammatory signaling. This escalating feedback loop provides novel targets and therapeutic strategies for the prevention of preterm labor and its consequences.

For successful embryo implantation, endometrial stromal cells must undergo functional and morphological changes, referred to as decidualization. However, the molecular mechanisms that regulate implantation and decidualization are not well defined. Here we demonstrate that the estradiol- and progesterone-regulated microRNA (miR)-200 family was markedly down-regulated in mouse endometrial stromal cells prior to implantation, whereas zinc finger E-box binding homeobox-1 and -2 and other known and predicted targets were up-regulated. Conversely, miR-200 was up-regulated during in vitro decidualization of human endometrial stromal cells. Knockdown of miR-200 negatively affected decidualization and prevented the mesenchymal-epithelial transition-like changes that accompanied decidual differentiation. Notably, superovulation of mice and humans altered miR-200 expression. Our findings suggest that hormonal alterations that accompany superovulation may negatively impact endometrial development and decidualization by causing aberrant miR-200 expression.

Lung alveolar type II cells uniquely synthesize surfactant, a developmentally regulated lipoprotein that is essential for breathing. Expression of the gene (SFTPA) encoding the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cyclic AMP (cAMP). cAMP induction of SP-A expression is repressed by transforming growth factor β (TGF-β) and by hypoxia. In this study, we found that expression of the microRNA 29 (miR-29) family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. miR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-β2, was coordinately decreased. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting their physiological relevance. This occurred through derepression of TGF-β signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor 1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells, and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in cotransfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-β signaling., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-β. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-β2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-β2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-β. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-β, serve important roles in the developmental regulation of type II cell differentiation and function in HFL., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The molecular mechanisms that maintain quiescence of the myometrium throughout most of pregnancy and promote its transformation to a highly coordinated contractile unit culminating in labor are complex and intertwined. During pregnancy, progesterone (P4) produced by the placenta and/or ovary serves a dominant role in maintaining myometrial quiescence by blocking proinflammatory response pathways and expression of so-called "contractile" genes. In the majority of placental mammals, increased uterine contractility near term is heralded by an increase in circulating estradiol-17β (E2) and/or increased estrogen receptor α (ERα) activity and a sharp decline in circulating P4 levels. However, in women, circulating levels of P4 and progesterone receptors (PR) in myometrium remain elevated throughout pregnancy and into labor. This has led to the concept that increased uterine contractility leading to term and preterm labor is mediated, in part, by a decline in PR function. The biochemical mechanisms for this decrease in PR function are also multifaceted and interwoven. In this paper, we focus on the molecular mechanisms that mediate myometrial quiescence and contractility and their regulation by the two central hormones of pregnancy, P4 and estradiol-17β. The integrative roles of microRNAs also are considered., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process.

Persistent hypoxia caused by shallow trophoblast invasion and poor placental perfusion may underlie the pathophysiology of preeclampsia, a leading cause of maternal and neonatal morbidity and mortality. Previously, we found that estrogen-related receptor γ (ERRγ) serves a critical and O2-dependent role in differentiation of human trophoblasts in culture and expression of tissue kallikrein and voltage-gated K(+) channels. In this study, we surprisingly observed that ERRγ expression was significantly increased in placentas from preeclamptic women compared with that in gestation-matched normotensive women. To further investigate a functional role for ERRγ during pregnancy, we analyzed ERRγ-deficient mice. Maternal systolic blood pressure was significantly reduced in pregnant ERRγ(+/-) females bred to ERRγ(+/-) males compared with that in wild-type (WT) mice and was markedly up-regulated by treatment of WT pregnant mice with the ERRγ agonist DY131. Placentas of ERRγ(+/-) mice manifested increased vascular endothelial growth factor A expression compared with that in WT mice. Notably, circulating levels of the antiangiogenic factor, soluble fms-like tyrosine kinase-1, were significantly reduced in ERRγ(+/-) pregnant mice as was serum aldosterone. These effects were associated with a decrease in maternal adrenal Cyp11b1 (steroid 11β-hydroxylase) and Cyp11b2 (aldosterone synthase) expression. In contrast, adrenal Cyp11b1 and Cyp11b2 mRNA were increased in pregnant WT mice treated with DY131. Moreover, chromatin immunoprecipitation and luciferase reporter assays identified Cyp11b2 as a transcriptional target of ERRγ. Collectively, these findings reveal a potential role of ERRγ in maternal blood pressure homeostasis during pregnancy and suggest that aberrant ERRγ expression may contribute to the pathogenesis of preeclampsia.

The maintenance of myometrial quiescence and initiation of contractility, which lead to parturition at term and preterm, involve a shifting equilibrium between anti-inflammatory and proinflammatory signalling pathways. Progesterone (P4), acting through the progesterone receptor (PR), has an essential and multifaceted role in the maintenance of myometrial quiescence. This effect of P4-PR signalling is mediated, in part, by its anti-inflammatory actions and capacity to repress the expression of genes that encode proinflammatory cytokines, such as IL-1 and IL-6, and contraction-associated proteins, such as OXTR, GJA1 and PTGS2. By contrast, increased expression of genes that ultimately lead to parturition is mediated by enhanced inflammatory and estradiol-17β (E2) and estrogen receptor α signalling, which reduce PR function, thus further intensifying the inflammatory response. To obtain a more complete understanding of the molecular events that underlie the transition of the pregnant myometrium from a refractory to a contractile state, the roles of microRNAs, their targets, and their transcriptional and hormonal regulation have been investigated. This article reviews the actions of the miR-200 family and their P4-regulated targets-the transcription factors ZEB1, ZEB2 and STAT5B-in the pregnant myometrium, as well as the role of miR-199a-3p and miR-214 and their mutual target PTGS2. The central role of ZEB1 as the mediator of the opposing actions of P4 and E2 on myometrial contractility will be highlighted.

Estrogen-related receptor γ (ERRγ) serves a critical O2-dependent regulatory role in the differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikrein (KLK1) and voltage-gated K(+) channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRγ and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, and KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRγ, expression of KLK1, KCNQ1, KCNE1, KCNE3, and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with short hairpin RNAs for endogenous ERRγ, KLK1, KCNQ1, KCNE1, and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements within the KLK1 and KCNE1 5'-flanking regions required for ERRγ-stimulated transcriptional activity. Binding of endogenous ERRγ to these ERRγ response elements increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced human chorionic gonadotropin secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ mediates O2-dependent expression of genes involved in human trophoblast differentiation, function, and vascular homeostasis.

Mononuclear cytotrophoblasts of the human placenta proliferate rapidly, subsequently fuse, and differentiate to form multinucleated syncytiotrophoblast with induction of aromatase (hCYP19A1) and chorionic gonadotropin (hCGβ) expression. Using microarray analysis, we identified members of the miR-17~92 cluster and its paralogs, miR-106a~363 and miR-106b~25, that are significantly downregulated upon syncytiotrophoblast differentiation. Interestingly, miR-19b and miR-106a directly targeted hCYP19A1 expression, while miR-19b also targeted human GCM1 (hGCM1), a transcription factor critical for mouse labyrinthine trophoblast development. Overexpression of these microRNAs (miRNAs) impaired syncytiotrophoblast differentiation. hGCM1 knockdown decreased hCYP19A1 and hCGβ expression, substantiating its important role in human trophoblast differentiation. Expression of the c-Myc proto-oncogene was increased in proliferating cytotrophoblasts compared to that in differentiated syncytiotrophoblast. Moreover, c-Myc overexpression upregulated miR-17~92 and inhibited hCYP19A1 and hCGβ expression. Binding of endogenous c-Myc to genomic regions upstream of the miR-17~92 and miR-106a~363 clusters in cytotrophoblasts dramatically decreased upon syncytiotrophoblast differentiation. Intriguingly, we observed higher levels of miR-106a and -19b and lower aromatase and hGCM1 expression in placentas from preeclamptic women than in placentas from gestation-matched normotensive women. Our findings reveal that c-Myc-regulated members of the miR-17~92 and miR-106a~363 clusters inhibit trophoblast differentiation by repressing hGCM1 and hCYP19A1 and suggest that aberrant regulation of these miRNAs may contribute to the pathogenesis of preeclampsia.

Previously we obtained compelling evidence that the fetus provides a critical signal for the initiation of term labor through developmental induction of surfactant protein (SP)-A expression by the fetal lung and secretion into amniotic fluid (AF). We proposed that interactions of AF macrophage (Mϕ) Toll-like receptors (TLRs) with SP-A, at term, or bacterial components, at preterm, result in their activation and migration to the pregnant uterus. Herein the timing of labor in wild-type (WT) C57BL/6 mice was compared with mice homozygous null for TLR2, SP-A, SP-D, or doubly deficient in SP-A and SP-D. Interestingly, TLR2(-/-) females manifested a significant (P < 0.001) delay in timing of labor compared with WT as well as reduced expression of the myometrial contraction-associated protein (CAP) gene, connexin-43, and Mϕ marker, F4/80, at 18.5 d postcoitum (dpc). Whereas in first pregnancies, SP-A(-/-), SP-D(-/-), and SP-A/D(-/-) females delivered at term (∼19.5 dpc), in second pregnancies, parturition was delayed by approximately 12 h in SP-A(-/-) (P = 0.07) and in SP-A/D(-/-) (P <0.001) females. Myometrium of SP-A/D(-/-) females expressed significantly lower levels of IL-1β, IL-6, and CAP genes, connexin-43, and oxytocin receptor at 18.5 dpc compared with WT. F4/80(+) AF Mϕs from TLR2(-/-) and SP-A/D(-/-) mice expressed significantly lower levels of both proinflammatory and antiinflammatory activation markers (e.g. IL-1β, IL-6, ARG1, YM1) compared with gestation-matched WT AF Mϕs. These novel findings suggest that the pulmonary collectins acting via TLR2 serve a modulatory role in the timing of labor; their relative impact may be dependent on parity.

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.

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.

The roles of progesterone (P(4)) and of progesterone receptor (PR) in development and pathogenesis of breast cancer remain unclear. In this study, we observed that treatment of T47D breast cancer cells with progestin antagonized effects of fetal bovine serum (FBS) to stimulate cell proliferation, whereas siRNA-mediated knockdown of endogenous PR abrogated progestin-mediated anti-proliferative effects. To begin to define mechanisms for the anti-proliferative action of P(4)/PR, we considered the role of MAPK phosphatase 1 (MKP-1/DUSP1), which catalyzes dephosphorylation and inactivation of MAPKs. Progestin treatment of T47D cells rapidly induced MKP-1 expression in a PR-dependent manner. Importantly, P(4) induction of MKP-1 was associated with reduced levels of phosphorylated ERK1/2, whereas siRNA knockdown of MKP-1 blocked progestin-mediated ERK1/2 dephosphorylation and repression of FBS-induced cell proliferation. The importance of PR in MKP-1 expression was supported by findings that MKP-1 and PR mRNA levels were significantly correlated in 30 human breast cancer cell lines. By contrast, no correlation was observed with the glucocorticoid receptor, a known regulator of MKP-1 in other cell types. ChIP and luciferase reporter assay findings suggest that PR acts in a ligand-dependent manner through binding to two progesterone response elements downstream of the MKP-1 transcription start site to up-regulate MKP-1 promoter activity. PR also interacts with two Sp1 sites just downstream of the transcription start site to increase MKP-1 expression. Collectively, these findings suggest that MKP-1 is a critical mediator of anti-proliferative and anti-inflammatory actions of PR in the breast.

Differentiation of human cytotrophoblasts to syncytiotrophoblast and the associated induction of aromatase/hCYP19 gene expression are dependent upon a critical O(2) tension; however, the underlying molecular mechanisms remain undefined. In this study, we provide compelling evidence that expression of the orphan nuclear receptor, estrogen-related receptor γ (ERRγ), is also O(2) dependent, induced during human syncytiotrophoblast differentiation, and plays an obligatory role in the induction of placenta-specific hCYP19I.1 gene expression. Treatment with the selective ERRγ agonist, DY131, or overexpression of ERRγ, stimulated hCYP19 expression in syncytiotrophoblast. Overexpression of ERRγ prevented effects of hypoxia to repress hCYP19 gene expression in cultured trophoblasts. Conversely, small interfering RNA-mediated knockdown of endogenous ERRγ in primary trophoblasts markedly inhibited hCYP19 expression. Promoter and site-directed mutagenesis studies in transfected placental cells identified a nuclear receptor element within placenta-specific hCYP19 promoter I.1 required for ERRγ-stimulated activity. Recruitment of endogenous ERRγ to the nuclear receptor element region in hCYP19 promoter during trophoblast differentiation, assessed by chromatin immunoprecipitation, was prevented by hypoxia. Deferoxamine-induced hypoxia-inducible factor-1α (HIF-1α) levels decreased ERRγ expression, whereas knockdown of endogenous HIF-1α prevented ERRγ suppression by hypoxia. Chromatin immunoprecipitation analysis of trophoblasts cultured in hypoxia revealed recruitment of HIF-1α to one of two putative hypoxia response elements in the ERRγ promoter, providing in vivo evidence of a direct HIF-1α involvement in ERRγ expression. Collectively, these novel findings identify ERRγ as an O(2)-dependent transcription factor and HIF-1α target gene that serves a critical role in the induction of hCYP19 expression during human trophoblast differentiation.

SP-A gene expression is developmentally regulated in fetal lung. Cyclic AMP (cAMP) induction of SP-A expression in human fetal lung type II cells is O(2) dependent and is mediated by increased binding of TTF-1/Nkx2.1 and NF-κB to a critical response element (TBE). This is associated with increased acetylation and decreased methylation of H3K9 at the TBE. Using chromatin immunoprecipitation analysis of fetal lung between 15.5 and 19.0 days of gestation, we observed that the developmental induction of SP-A was associated with increased recruitment of TTF-1, NF-κB, PCAF, and CBP, as well as enhanced acetylation and decreased methylation of histone H3K9 at the TBE. Importantly, expression and TBE binding of the H3K9 methyltransferases, Suv39h1 and Suv39h2, was inversely correlated with the developmental upregulation of SP-A. In human fetal lung epithelial cells, Suv39H1 and Suv39H2 mRNA levels declined with cAMP induction of SP-A. Moreover, hypoxia, which inhibits cAMP stimulation of SP-A, markedly increased Suv39h1 and Suv39h2 binding to the TBE. Finally, short hairpin RNA knockdown of Suv39H1 or Suv39H2 in fetal lung epithelial cells repressed H3K9 methylation and greatly enhanced SP-A expression. Collectively, our findings suggest that Suv39H1 and Suv39H2 are key hypoxia-induced methyltransferases; their decline in fetal lung during late gestation is critical for epigenetic changes resulting in the developmental induction of SP-A.

Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, whereas spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and have a negative impact on PR function. In this study, we uncovered a previously undescribed regulatory pathway whereby micro-RNAs (miRNAs) serve as hormonally modulated and conserved mediators of contraction-associated genes in the pregnant uterus in the mouse and human. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans as well as two coordinately down-regulated targets, zinc finger E-box binding homeobox proteins ZEB1 and ZEB2, which act as transcriptional repressors. We also observed up-regulation of the miR-200 family and down-regulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. We further demonstrated that ZEB1 is directly up-regulated by the action of progesterone (P(4))/PR at the ZEB1 promoter. Excitingly, we observed that ZEB1 and ZEB2 inhibit expression of the contraction-associated genes, oxytocin receptor and connexin-43, and block oxytocin-induced contractility in human myometrial cells. Together, these findings implicate the miR-200 family and their targets, ZEB1 and ZEB2, as unique P(4)/PR-mediated regulators of uterine quiescence and contractility during pregnancy and labor and shed light on the molecular mechanisms involved in preterm birth.

Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

Mechanisms underlying the initiation of parturition remain unclear. Throughout most of pregnancy, uterine quiescence is maintained by elevated progesterone acting through progesterone receptor (PR). Although in most mammals, parturition is associated with a marked decline in maternal progesterone, in humans, circulating progesterone and uterine PR remain elevated throughout pregnancy, suggesting a critical role for functional PR inactivation in the initiation of labor. Both term and preterm labor in humans and rodents are associated with an inflammatory response. In preterm labor, intraamniotic infection likely provides the stimulus for increased amniotic fluid interleukins and migration of inflammatory cells into the uterus and cervix. However, at term, the stimulus for this inflammatory response is unknown. Increasing evidence suggests that the developing fetus may produce physical and hormonal signals that stimulate macrophage migration to the uterus, with release of cytokines and activation of inflammatory transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), which also is activated by myometrial stretch. We postulate that the increased inflammatory response and NF-kappaB activation promote uterine contractility via 1) direct activation of contractile genes (e.g. COX-2, oxytocin receptor, and connexin 43) and 2) impairment of the capacity of PR to mediate uterine quiescence. PR function near term may be compromised by direct interaction with NF-kappaB, altered expression of PR coregulators, increased metabolism of progesterone within the cervix and myometrium, and increased expression of inhibitory PR isoforms. Alternatively, we propose that uterine quiescence during pregnancy is regulated, in part, by PR antagonism of the inflammatory response.

A 246-bp region upstream of placenta-specific exon I.1 of the human aromatase (hCYP19) gene mediates placenta-specific, developmental, and O(2) regulation of expression. In this study, trophoblast differentiation and associated induction of CYP19 expression were prevented when cytotrophoblasts were cultured in phenol red-free medium containing charcoal-stripped serum or with the estrogen receptor (ER) antagonist, ICI 182,780, suggesting a stimulatory role of estrogen/ER. ERalpha protein was expressed in human trophoblasts and increased during syncytiotrophoblast differentiation, whereas ERbeta was undetectable. Mutational analysis revealed that an estrogen response element-like sequence (ERE-LS) at -208 bp is required for inductive effects of estradiol/ERalpha on hCYP19I.1 promoter activity in transfected COS-7 cells. Increased binding of syncytiotrophoblast compared with cytotrophoblast nuclear proteins to the ERE-LS was observed in vitro; however, ERalpha antibodies failed to supershift the complex and in vitro-transcribed/translated ERalpha did not bind. Nonetheless, chromatin immunoprecipitation assays in cultured trophoblasts revealed recruitment of endogenous ERalpha to the -255- to -155-bp region containing the ERE-LS before induction of hCYP19 expression; this was inhibited by ICI 182,780. Chromatin immunoprecipitation also revealed increased acetylated histone H3(K9/14) and decreased methylated histone H3(K9) associated with this region during trophoblast differentiation. These modifications were prevented when trophoblasts were incubated with ICI 182,780, suggesting that ERalpha recruitment to the -255- to -155-bp region promotes histone modifications leading to increased hCYP19 transcription. Thus, during trophoblast differentiation, estrogen/ERalpha exerts a positive feedback role, which promotes permissive histone modifications that are associated with induction of hCYP19 gene transcription.

Estrogen-related receptor (ERRalpha) plays a critical role in basal and cAMP-induced expression of the human surfactant protein-A (SP-A) gene in lung type II cells through direct binding to an ERR response element (ERRE, 5'-TGACCTTA-3') within its 5'-flanking region. Furthermore, protein kinase A (PKA) up-regulates ERRalpha activation of the hSP-A promoter. In the present study, using cultured human fetal lung type II cells, we observed that cAMP enhanced ERRalpha phosphorylation and nuclear expression levels. cAMP/PKA stimulation of ERRalpha activation of the SP-A promoter was blocked by the PKA inhibitor, H89, whereas the MAPK P38 inhibitor, SB203580, and the MAPK kinase inhibitor, PD98059, had negligible to modest effects. This suggests that cAMP acts selectively through PKA to increase ERRalpha transcriptional activity. Of several coactivators tested, steroid receptor coactivator 2 (SRC-2) had the most pronounced effect to increase ERRalpha transcriptional activity at the SP-A promoter; this was enhanced by cotransfection with PKA catalytic subunit (PKAcat). Interestingly, SRC-2, ERRalpha, and PKAcat in type II cell nuclear extracts interacted at the ERRE; this was enhanced by cAMP and inhibited by H89. cAMP increased in vivo binding of PKAcat and SRC-2 to the ERRE genomic region in lung type II cells. In mutagenesis studies, three serines (S87, S114, and S277) were found to be critical for PKA and SRC-2 induction of ERRalpha transcriptional activity. Collectively, these findings indicate that cAMP/PKA signaling enhances ERRalpha phosphorylation and nuclear localization, recruitment to the SP-A promoter, and interaction with PKAcat and SRC-2, resulting in the up-regulation of SP-A gene transcription.

Context: Up-regulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity may enhance their survival via local estrogen synthesis, which may lead to endometriosis. The factors that mediate induction of aromatase in the endometrium are not well defined, but increased expression of steroidogenic factor (SF)-1 may play a role., Objective: The objective of the study was to determine whether androstenedione (A4), the predominant sex steroid in peritoneal fluid, regulates endometrial aromatase expression., Design: This was a cell/tissue culture study., Setting: The study was conducted at an academic center., Methods: Quantitative real-time PCR, HPLC, and chromatin immunoprecipitation were used in this study., Results: Treatment of cultured human endometrial explants and stromal cells with A4 (10 nm) significantly up-regulated expression of aromatase mRNA transcripts containing exon IIa at their 5'-ends. In endometrial stromal cells and the human endometrial surface epithelial (HES) cell line, induction of aromatase mRNA by A4 was associated with increased expression of SF-1. In HES cells, tritiated A4 was metabolized to estradiol, testosterone (T), dihydrotestosterone, and androstanediol. Both estradiol and T, but not nonaromatizable androgens, up-regulated aromatase and SF-1 mRNA in HES cells. Chromatin immunoprecipitation revealed that A4 enhanced recruitment of SF-1 to its response element (-136 bp) upstream of CYP19 exon IIa. This, together with the findings that both estrogen receptor antagonist, ICI 182,780, and aromatase inhibitor, fadrozole, suppressed A4 and T induction of aromatase and SF-1 mRNA, indicates that the inductive effects of A4 and T are mediated by their conversion to estrogens., Conclusions: Exposure of endometrial cells to A4 may enhance CYP19 gene expression through its aromatization to estrogens.

Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara cells, and is developmentally and hormonally regulated in fetal lung. Using transfected human fetal type II cells, we previously observed that approximately 300 bp of 5'-flanking DNA mediated cAMP and interleukin-1 (IL-1) stimulation and dexamethasone (Dex) inhibition of hSP-A2 promoter activity. This region contains response elements for estrogen-related receptor alpha element (ERRE, -241 bp), thyroid transcription factor (TTF)-1/Nkx2.1 (TTF-binding protein, -171 bp), upstream stimulatory factor 1/2 (E-box, -80 bp), and stimulatory protein (Sp) 1 (G/T-box, -62 bp), which are essential for basal and cAMP induction of hSP-A2 expression. To define genomic regions necessary for developmental, hormonal, and tissue-specific regulation of hSP-A2 expression in vivo, we analyzed transgenic mice carrying hGH reporter genes comprised of 313 bp of hSP-A2 gene 5'-flanking DNA +/- mutation in the TBE or 175 bp of 5'-flanking DNA, containing TBE, E-box and G/T-box, but lacking ERRE. Transgenes containing 313 or 175 bp of hSP-A2 5'-flanking DNA were expressed in a lung cell-specific manner and developmentally regulated in concert with the endogenous mouse SP-A gene. In cultured lung explants from hSP-A(-313):hGH transgenic fetal mice, cAMP and IL-1 induced and Dex inhibited transgene expression. However, the 175-bp hSP-A2 genomic region was insufficient to mediate hormonal regulation of hSP-A2 promoter activity. The finding that expression of the hSP-A(-313TBEmut):hGH transgene was essentially undetectable in fetal lung and was not hormonally regulated in transgenic fetal lung explants underscores the critical importance of the TBE in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression.