Decline in corepressor CNOT1 in the pregnant myometrium near term impairs progesterone receptor function and increases contractile gene expression.

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.

Arginase 1 and L-arginine coordinate fetal lung development and the initiation of labor in mice.

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.

IL-1ß mediates the induction of immune checkpoint regulators IDO1 and PD-L1 in lung adenocarcinoma cells.

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.

Role of NRF2 in immune modulator expression in developing lung.

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.

Decreased 11ß-hydroxysteroid dehydrogenase 1 in lungs of steroid receptor coactivator (Src)-1/-2 double-deficient fetal mice is caused by impaired glucocorticoid and cytokine signaling.

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.

Primate-specific miR-515 family members inhibit key genes in human trophoblast differentiation and are upregulated in preeclampsia.

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.

The transcriptional repressor GATAD2B mediates progesterone receptor suppression of myometrial contractile gene expression.

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.

The miR-200 family and its targets regulate type II cell differentiation in human fetal lung.

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.

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

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

miR-200 family and targets, ZEB1 and ZEB2, modulate uterine quiescence and contractility during pregnancy and labor.

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.

Progesterone receptor inhibits proliferation of human breast cancer cells via induction of MAPK phosphatase 1 (MKP-1/DUSP1).

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.

NRF2 Serves a Critical Role in Regulation of Immune Checkpoint Proteins (ICPs) During Trophoblast Differentiation.

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.

Surfactant protein A2 mutations associated with pulmonary fibrosis lead to protein instability and endoplasmic reticulum stress.

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.

Permissive effects of oxygen on cyclic AMP and interleukin-1 stimulation of surfactant protein A gene expression are mediated by epigenetic mechanisms.

Surfactant protein A (SP-A) is important for immune defense within the alveolus. Cyclic AMP (cAMP) stimulation of SP-A expression in lung type II cells is O(2) dependent and mediated by increased phosphorylation and binding of thyroid transcription factor 1 (TTF-1) to an upstream response element (TTF-1-binding element [TBE]). Interleukin-1 (IL-1) stimulation of SP-A expression is mediated by NF-kappaB (p65/p50) activation and increased binding to the TBE. In this study, we found that O(2) also was permissive for IL-1 induction of SP-A expression and for cAMP and IL-1 stimulation of type II cell nuclear protein binding to the TBE. Using chromatin immunoprecipitation, we observed that when type II cells were cultured in 20% O(2), cAMP and IL-1 stimulated the recruitment of TTF-1, p65, CBP, and steroid receptor coactivator 1 to the TBE region of the SP-A promoter and increased local acetylation of histone H3; these effects were prevented by hypoxia. Hypoxia markedly reduced global levels of CBP and acetylated histone H3 and increased the expression of histone deacetylases. Furthermore, hypoxia caused a global increase in histone H3 dimethylated on Lys9 and increased the association of dimethyl histone H3 with the SP-A promoter. These results, together with findings that the histone deacetylase inhibitor trichostatin A and the methyltransferase inhibitor 5'-deoxy(5'-methylthio)adenosine markedly enhanced SP-A expression in lung type II cells, suggest that increased O(2) availability to type II cells late in gestation causes epigenetic changes that permit access of TTF-1 and NF-kappaB to the SP-A promoter. The binding of these transcription factors facilitates the recruitment of coactivators, resulting in the further opening of the chromatin structure and activation of SP-A transcription.

Glucocorticoid/glucocorticoid receptor inhibition of surfactant protein-A (SP-A) gene expression in lung type II cells is mediated by repressive changes in histone modification at the SP-A promoter.

Surfactant protein-A (SP-A) gene expression in human fetal lung type II cells is stimulated by cAMP and IL-1 and is inhibited by glucocorticoids. cAMP/IL-1 stimulation of SP-A expression is mediated by increased binding of thyroid transcription factor-1 and nuclear factor (NF)-kappaB to the TTF-1-binding element (TBE) in the SP-A promoter. This is associated with decreased expression of histone deacetylases (HDACs), increased recruitment of coactivators, and enhanced acetylation of histone H3 (K9,14) at the TBE. In the present study, endogenous glucocorticoid receptor (GR) was found to interact with thyroid transcription factor-1 and NF-kappaB p65 at the TBE. GR knockdown enhanced SP-A expression in type II cells cultured in serum-free medium, suggesting a ligand-independent inhibitory role of endogenous GR. Furthermore, use of chromatin immunoprecipitation revealed that dexamethasone (Dex) treatment of fetal lung type II cells increased recruitment of endogenous GR and HDACs-1 and -2 and blocked cAMP-induced binding of inhibitor of kappaB kinase-alpha (IKKalpha) to the TBE region. Accordingly, Dex reduced basal and blocked cAMP-stimulated levels of acetylated (K9,14) and phosphorylated (S10) histone H3 at the TBE. Dex also increased TBE binding of dimethylated histone H3 (K9) and of heterochromatin protein 1alpha. Thus, Dex increases interaction of GR with the complex of proteins at the TBE. This facilitates recruitment of HDACs and causes a local decline in basal and cAMP-induced histone H3 phosphorylation and acetylation and an associated increase in H3-K9 dimethylation and binding of heterochromatin protein 1alpha. Collectively, these events may culminate in the closing of chromatin structure surrounding the SP-A gene and inhibition of its transcription.

Progesterone receptor inhibits aromatase and inflammatory response pathways in breast cancer cells via ligand-dependent and ligand-independent mechanisms.

Aromatase (product of CYP19 gene), the critical enzyme in estrogen biosynthesis, is up-regulated in 70% of all breast cancers and is highly correlated with cyclooxygenase 2 (COX-2), the rate-determining enzyme in prostanoid biosynthesis. Expression of COX-2 also is correlated with the oncogene HER-2/neu. The efficacy of current endocrine therapies for breast cancer is predicted only if the tumor contains significant amounts of estrogen receptor. Because the progesterone receptor (PR) is an estrogen-induced target gene, it has been suggested that its presence may serve as an indicator of estrogen receptor functional capacity and the differentiation state of the tumor. In the present study, we tested the hypothesis that PR serves a crucial protective role by antagonizing inflammatory response pathways in the breast. We observed that progesterone antagonized the stimulatory effects of cAMP and IL-1beta on aromatase, COX-2, and HER-2/neu expression in T47D breast cancer cells. These actions of progesterone were associated with increased expression of the nuclear factor-kappaB inhibitor, IkappaBalpha. In 28 breast cancer cell lines, IkappaBalpha expression was positively correlated with PR mRNA levels; overexpression of a phosphorylation-defective mutant of IkappaBalpha inhibited expression of aromatase, COX-2, and HER-2/neu. Moreover, in breast cancer cell lines cultured in the absence of progesterone, up-regulation of endogenous PR caused decreased expression of aromatase, COX-2, and HER-2/neu expression, whereas down-regulation of endogenous PR resulted in a marked induction of aromatase and HER-2/neu mRNA. Collectively, these findings suggest that PR plays an important antiinflammatory role in breast cancer cells via ligand-dependent and ligand-independent mechanisms.

Multifactorial Regulation of Myometrial Contractility During Pregnancy and Parturition.

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.

Human Trophoblast Differentiation Is Associated With Profound Gene Regulatory and Epigenetic Changes.

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.

Inflammatory status influences aromatase and steroid receptor expression in endometriosis.

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.

Androstenedione up-regulation of endometrial aromatase expression via local conversion to estrogen: potential relevance to the pathogenesis of endometriosis.

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.