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Progesterone (P4), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P4/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 P4/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 P4-treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P4 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 P4/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.
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Miométrio , Regiões Promotoras Genéticas , Receptores de Progesterona , Animais , Feminino , Humanos , Camundongos , Gravidez , Conexina 43/metabolismo , Conexina 43/genética , Regulação da Expressão Gênica , Miométrio/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Contração Uterina/metabolismo , Contração Uterina/genéticaRESUMO
INTRODUCTION: Inflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1ß is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1ß compared to healthy individual. Moreover, IL-1ß 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-1ß levels and is likely caused by IL-1ß. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1ß 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-1ß is a common upstream regulator of immune checkpoint regulators. METHODS: To determine whether IL-1ß 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-1ß -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1ß treated cells. The levels of Trp and Kyn in the IL-1ß-treated cells and media were measured by mass spectrometry. RESULTS: Upon IL-1ß 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-1ß, aligning with the heightened IDO1 expression. Remarkably, IL-1ß also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1ß 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-1ß signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion. Video Abstract.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/metabolismo , Antígeno B7-H1/metabolismo , Cinurenina/metabolismo , Neoplasias Pulmonares/patologia , RNA Mensageiro , Triptofano , Microambiente TumoralRESUMO
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/EBPß 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.
Assuntos
Arginase , Pulmão , Animais , Humanos , Camundongos , Arginase/genética , Arginase/metabolismo , Arginina/metabolismo , Desenvolvimento Fetal , Feto/metabolismo , Camundongos KnockoutRESUMO
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/EBPß, 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/EBPß, aromatase (CYP19A1; SynT differentiation marker), and ICPs were blocked. NRF2 knockdown also prevented induction of aromatase, C/EBPß 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/ebpß, 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.
Assuntos
Aromatase , Trofoblastos , Animais , Aromatase/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Feminino , Proteínas de Checkpoint Imunológico , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , PPAR gama/metabolismo , Gravidez , Trofoblastos/metabolismoRESUMO
After birth, the alveolar epithelium is exposed to environmental pathogens and high O2 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/EBPß 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/EBPß, 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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/imunologia , Pulmão , Fator 2 Relacionado a NF-E2 , Animais , Feminino , Humanos , Pulmão/embriologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos ICR , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/imunologiaRESUMO
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 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which catalyzes conversion of inactive 11-dehydrocorticosterone to the glucocorticoid receptor (GR) ligand, corticosterone. Glucocorticoids were reported to upregulate 11ß-HSD1 expression in various cell types via induction of C/EBP transcription factors. We observed that C/ebpα and C/ebpß mRNA and protein were markedly reduced in Src-1/-2 double-deficient (Src-1/-2d/d ) fetal lungs, compared to WT. Moreover, glucocorticoid induction of 11ß-hsd1, C/ebpα and C/ebpß in cultured MFL epithelial cells was prevented by the SRC family inhibitor, SI-2. Cytokines also contribute to the induction of 11ß-HSD1. Expression of IL-1ß and TNFα, which dramatically increased toward term in lungs of WT fetuses, was markedly reduced in Src-1/-2d/d fetal lungs. Our collective findings suggest that impaired lung development and surfactant synthesis in Src-1/-2d/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 11ß-HSD1 expression and glucocorticoid signaling within the fetal lung, causing a break in the glucocorticoid-induced positive feedforward loop.
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11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Citocinas/metabolismo , Feto/metabolismo , Glucocorticoides/metabolismo , Pulmão/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica/fisiologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The steroid hormones progesterone (P4) and estradiol-17ß (E2), 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 P4 and its nuclear receptor (PR) maintain myometrial quiescence during pregnancy, and for the decline in P4/PR and increase in E2/estrogen receptor (ER) function leading to parturition, are multifaceted, cooperative, and redundant. These actions of P4/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 P4 levels remain elevated throughout most of pregnancy and decline precipitously near term. By contrast, in humans, circulating P4 levels and myometrial PR levels remain elevated throughout pregnancy and into labor. However, even in rodents, wherein P4 levels decline near term, P4 levels remain higher than the Kd for PR binding. Thus, parturition is initiated in all species by a series of molecular events that antagonize the P4/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 P4 metabolizing enzymes; increased expression of truncated/inhibitory PR isoforms; altered expression of PR coactivators and corepressors. This article will review various mechanisms whereby P4 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 P4- and E2-regulated miRNAs in the regulation and integration of these mechanisms will also be considered.
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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.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Trofoblastos/citologia , Diferenciação Celular , Fusão Celular , Células Cultivadas , Feminino , Histonas/metabolismo , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Placenta/fisiologia , Gravidez , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Trofoblastos/metabolismoRESUMO
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/EBPß, 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/EBPß 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.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Fatores de Transcrição/genética , AMP Cíclico/metabolismo , Regulação para Baixo , Humanos , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
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 GSK3ß and AXIN2, inhibitors of WNT/ß-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-GSK3ß, 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, GSK3ß, 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/EBPß 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/EBPß, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.
Assuntos
Diferenciação Celular/genética , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/genética , Trofoblastos/metabolismo , Aromatase/genética , Proteína Axina/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Imunoprecipitação da Cromatina , Feminino , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Oxirredução , PPAR gama/metabolismo , Placenta/citologia , Complexo Repressor Polycomb 2/metabolismo , Reação em Cadeia da Polimerase , Gravidez , Trofoblastos/citologia , Via de Sinalização WntRESUMO
The mechanisms whereby progesterone (P4), 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 (PRWT), we found that P4 significantly inhibited IL-1ß induction of the NF-κB target genes, COX-2 and IL-8 P4-PRWT 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 (PRmDBD), P4-mediated transrepression was significantly reduced, suggesting a critical role of the PR DBD. ChIP analysis of hTERT-HM cells stably expressing PRWT or PRmDBD revealed that P4 treatment caused equivalent recruitment of PRWT and PRmDBD 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 PRWT but poorly with PRmDBD P4 treatment of PRWT hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P4-PRWT 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 P4-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.
Assuntos
Regulação para Baixo , Fatores de Transcrição GATA/metabolismo , Miométrio/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Repressoras/metabolismo , Contração Uterina/genética , Animais , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Células HEK293 , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Interleucina-8/metabolismo , Camundongos , Miométrio/efeitos dos fármacos , Progesterona/farmacologia , Receptores de Progesterona/agonistasRESUMO
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.
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Troca Materno-Fetal , Parto , Transdução de Sinais , Animais , Feminino , Humanos , Gravidez , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Fatores de TempoRESUMO
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.
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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.
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Endométrio/citologia , Endométrio/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Endométrio/crescimento & desenvolvimento , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Immunoblotting , Técnicas In Vitro , Camundongos , MicroRNAs/genética , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/citologia , Células Estromais/metabolismo , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
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-1ß-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-1ß-induced contraction of human myometrial cells. Notably, miR-181a directly targeted ERα and decreased its expression, whereas estradiol-17ß reciprocally inhibited expression of mature miR-181a in myometrial cells. CONCLUSIONS: Thus, increased estradiol-17ß/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.
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Citocinas/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Inflamação/metabolismo , Trabalho de Parto/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Miométrio/metabolismo , Adulto , Animais , Técnicas de Cultura de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Transdução de Sinais , Regulação para CimaRESUMO
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 ß (TGF-ß) 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-ß2, 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-ß 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-ß signaling.
Assuntos
Células Epiteliais Alveolares/citologia , Pulmão/embriologia , MicroRNAs/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Células A549 , Animais , Diferenciação Celular , Células Cultivadas , AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Pulmão/citologia , Camundongos , Regiões Promotoras Genéticas , Transdução de Sinais , Fator Nuclear 1 de Tireoide , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
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-17ß (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-17ß. The integrative roles of microRNAs also are considered.
Assuntos
Miométrio/fisiologia , Parto/fisiologia , Animais , Estradiol/fisiologia , Feminino , Humanos , MicroRNAs/fisiologia , Gravidez , Progesterona/fisiologia , Contração Uterina/fisiologiaRESUMO
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-ß. 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-ß2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-ß2 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-ß. 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-ß, serve important roles in the developmental regulation of type II cell differentiation and function in HFL.
Assuntos
Células Epiteliais Alveolares/metabolismo , Diferenciação Celular/fisiologia , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Idade Gestacional , MicroRNAs/biossíntese , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Epiteliais Alveolares/citologia , Proteínas de Ligação a DNA/metabolismo , Feto/citologia , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
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.
Assuntos
Troca Materno-Fetal/fisiologia , Coativador 1 de Receptor Nuclear/fisiologia , Coativador 2 de Receptor Nuclear/fisiologia , Parto/fisiologia , 1-Acilglicerofosfocolina O-Aciltransferase/deficiência , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Animais , Feminino , Maturidade dos Órgãos Fetais , Heterozigoto , Pulmão/embriologia , Pulmão/fisiologia , Luteólise , Masculino , Troca Materno-Fetal/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Coativador 1 de Receptor Nuclear/deficiência , Coativador 1 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/deficiência , Coativador 2 de Receptor Nuclear/genética , Fator de Ativação de Plaquetas/deficiência , Gravidez , Regiões Promotoras Genéticas , Proteína A Associada a Surfactante Pulmonar/deficiência , Transdução de Sinais , Ativação Transcricional , Útero/fisiologiaRESUMO
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 11ß-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.