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Embryo implantation and processes of early pregnancy occur in a low-oxygen tissue environment controlled by hypoxia-inducible transcription factors of the hypoxia inducible factor (Hif) family. Hif2α has emerged as a pivotal factor in mediating uterine adaptation to hypoxia during early pregnancy. In mice, endometrial stromal Hif2α regulates the expression of the vesicular trafficking protein Rab27b, which in turn controls the release of both secretory granules and extracellular vesicles (EVs). These EVs harbor a variety of cargoes that control essential functional steps during pregnancy. In both mice and humans, EVs secreted by decidualizing stromal cells are taken up by stromal, endothelial, and trophoblast cells to support decidualization, blood vessel formation, and trophoblast development in the uterine milieu during early pregnancy. In mice, embryonic Hifs are essential for placental development. In humans, Hifs intrinsic to the trophoblast precursor cells are critical for their differentiation to the extravillous trophoblast (EVT) lineage. During the process of implantation, the embryo first attaches to the uterine epithelium and then invades the underlying stroma, resulting in the transformation of the stroma into a secretory tissue that surrounds the embryo. An intricate dialogue allows the developing embryo and the maternal tissue to be in constant communication with each other. In many mammals, including humans, embryo implantation and early pregnancy events take place in a low-oxygen environment regulated by hypoxia-inducible transcription factors. The mechanisms by which maternal and embryonic tissue compartments adapt to hypoxia are essential for the success of pregnancy outcomes. In this review we highlight recent work describing signaling pathways that operate in the hypoxic uterus to facilitate embryo implantation and promote the successful establishment of pregnancy. [ABSTRACT FROM AUTHOR]

Tamoxifen, a breast cancer therapeutic, is a tissue-selective estrogen receptor modulator (SERM), which acts as an antiestrogen in the mammary tissue and displays estrogenic activity in other tissues such as bone and uterus. In order to understand the mechanisms underlying the antiestrogenic effect of this prototype SERM, we performed an analysis of the cofactors that interact with ER complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7. Employing chromatin immunoprecipitation (ChIP), we observed that treatment with OHT rapidly induces the binding of ERα to the E-responsive promoter regions of pS2 and c-myc genes. Promoter-bound OHT-complexed ERa coordinately recruited the components of a multiprotein complex containing the corepressor NCoR, histone deacetylase 3 (HDAC3), and a WD40-repeat protein TBL1. Surprisingly, the OHT-complexed ERα also recruited a chromatin-remodeling NuRD complex in which histone deacetylase 1 (HDAC1) is associated with several polypeptides including metastasis-associated protein ½ (MTA1/ 2), and SWI2/SNF2-related ATPase Mi2. Kinetic studies revealed that following OHT addition the recruitment of these HDAC complexes to pS2 or the c-myc promoter occurs in a sequential manner; the NCoR-HDAC3 complex is recruited earlier than the NuRD complex. Serial ChIP experiments indicated that the ER-NCoR-HDAC3 and ERNuRD complexes are distinct, and they do not occupy the target gene promoter simultaneously. We also established a close temporal link between the appearance of the HDAC complexes at the E-responsive regions of pS2 and c-myc promoters, local hypoacetylation of specific lysine residues in N-terminal tails of histones H3 and H4, and disappearance of RNA polymerase II from the target gene loci. Collectively, our studies indicated that transcriptional repression by tamoxifen-bound ER at E-regulated gene promoters involves a dynamic interplay of multiple distinct chromatin-modifying/remodeling complexes. [ABSTRACT FROM AUTHOR]

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T. lymphocyte antigen-2β (CTLA2β), a cysteine protease inhibitor, is expressed during PRinduced decidualization. The spatio-temporal expression of CTLA-2β mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2β expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2β was, therefore, dependent on PR as well as other onknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2β, we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2β helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2β in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast. [ABSTRACT FROM AUTHOR]

Phthalates are synthetic chemicals widely used as plasticizers and stabilizers in various consumer products. Because of the extensive production and use of phthalates, humans are exposed to these chemicals daily. While most studies focus on a single phthalate, humans are exposed to a mixture of phthalates on a regular basis. The impact of continuous exposure to phthalate mixture on uterus is largely unknown. Thus, we conducted studies in which adult female mice were exposed for 6 months to 0.15 ppm and 1.5 ppm of a mixture of phthalates via chow ad libitum. Our studies revealed that consumption of phthalate mixture at 0.15 ppm and 1.5 ppm for 6 months led to a significant increase in the thickness of the myometrial layer compared to control. Further investigation employing RNA-sequencing revealed an elevated transforming growth factor beta (TGF-β) signaling in the uteri of mice fed with phthalate mixture. TGF-β signaling is associated with the development of fibrosis, a consequence of excessive accumulation of extracellular matrix components, such as collagen fibers in a tissue. Consistent with this observation, we found a higher incidence of collagen deposition in uteri of mice exposed to phthalate mixture compared to unexposed controls. Second Harmonic Generation (SHG) imaging showed disorganized collagen fibers and nanoindentation indicated a local increase in uterine stiffness upon exposure to phthalate mixture. Collectively, our results demonstrate that chronic exposure to phthalate mixture can have adverse effects on uterine homeostasis. • Phthalate mixture exposure promotes uterine fibrosis. • Phthalate mixture exposure enhances TGF beta signaling. • Phthalate mixture exposure increases uterine stiffness. [ABSTRACT FROM AUTHOR]

The peptide hormone prolactin (PRL) and certain members of the epidermal growth factor (EGF) family play central roles in mammary gland development and physiology, and their dysregulation has been implicated in mammary tumorigenesis. Our recent studies have revealed that the CUB and zona pellucidalike domain-containing protein 1 (CUZD1) is a critical factor for PRL-mediated activation of the transcription factor STAT5 in mouse mammary epithelium. Of note, CUZD1 controls production of a specific subset of the EGF family growth factors and consequent activation of their receptors. Here, we found that consistent with this finding, CUZD1 overexpression in nontransformed mammary epithelial HC11 cells increases their proliferation and induces tumorigenic characteristics in these cells. When introduced orthotopically in mouse mammary glands, these cells formed adenocarcinomas, exhibiting elevated levels of STAT5 phosphorylation and activation of the EGF signaling pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRLinduced tumor cell proliferation in vitro. Pimozide administration to mice also suppressed CUZD1-driven mammary tumorigenesis in vivo. Analysis of human MCF7 breast cancer cells indicated that CUZD1 controls the production of the same subset of EGF family members in these cells as in the mouse. Moreover, pimozide treatment reduced the proliferation of these cancer cells. Collectively, these findings indicate that overexpression of CUZD1, a regulator of growth factor pathways controlled by PRL and STAT5, promotes mammary tumorigenesis. Blockade of the STAT5 signaling pathway downstream of CUZD1 may offer a therapeutic strategy for managing these breast tumors. [ABSTRACT FROM AUTHOR]

Di-isononyl phthalate (DiNP), an endocrine-disrupting chemical, is found in numerous consumer products and human exposure to this phthalate is becoming inevitable. The impact of DiNP exposure on the establishment and maintenance of pregnancy remains largely unknown. Thus, we conducted studies in which pregnant mice were exposed to an environmentally relevant dose (20 µg/kg BW/day) of DiNP on days 1–7 of gestation, then analyzed the effects of this exposure on pregnancy outcome. Our studies revealed that exposure to DiNP during this window led to fetal loss towards the end of gestation. Further studies showed that, although embryos were able to attach to the uterus, implantation sites in DiNP-exposed uteri exhibited impaired differentiation of stromal cells to decidual cells and an underdeveloped angiogenic network in the decidual bed. We also found that exposure to this phthalate has a significant effect on trophoblast differentiation and causes disorganization of the placental layers. The labyrinth was significantly reduced, resulting in compromised expression of nutrient transporters in the placentas of mice exposed to DiNP. These placental defects in DiNP-exposed females were the cause of fetal loss during the later stages of gestation. • DiNP exposure impacts uterine angiogenesis. • DiNP exposure affects placentation. • DiNP exposure affects fetal growth. [ABSTRACT FROM AUTHOR]

Concerted actions of estrogen and progesterone via their cognate receptors orchestrate changes in the uterine tissue, regulating implantation during early pregnancy. The uterine stromal cells undergo steroid-dependent differentiation into morphologically and functionally distinct decidual cells, which support embryonic growth and survival. The hormone-regulated pathways underlying this unique cellular transformation are not fully understood. Previous studies in the mouse revealed that, following embryo attachment, de novo synthesis of estrogen by the decidual cells is critical for stromal differentiation. In this study we report that Fos-related antigen 1 (FRA-1), a member of the Fos family of transcription factors, is a downstream target of regulation by intrauterine estrogen. FRA-1 expression was localized in the differentiating uterine stromal cells surrounding the implanted embryo. Attenuation of estrogen receptor (Esr1) expression by siRNA mediated silencing in primary uterine stromal cells suppressed FRA-1 expression. Furthermore, chromatin immunoprecipitation demonstrated direct recruitment of ESR1 to an estrogen response element in the Fra-1 promoter. Down-regulation of Fra-1 expression during in vitro decidualization blocked stromal differentiation and resulted in a marked decrease in stromal cell migration. Interestingly, FRA-1 controls the expression of matrix metalloproteinases MMP9 and MMP13, which are critical modulators of stromal extracellular matrix remodeling. Collectively, these results suggest that FRA-1, induced in response to estrogen signaling via ESR1, is a key regulator of stromal differentiation and remodeling during early pregnancy. [ABSTRACT FROM AUTHOR]

Abstract: Context: Uterine decidualization is critical to embryonic implantation and sustained pregnancy. Objective: To evaluate the role of gap junction intercellular communications and connexin (Cx) proteins in the morphological and biochemical differentiation of decidualized human endometrial stromal cell (ESC) cultures. Design: Translational cell biological study. Setting: Academic medical center. Patients: Endometrial tissue was provided by five healthy reproductive age women on no hormonal medication, undergoing laparoscopy in the early proliferative phase of the menstrual cycle. Interventions: Endometrial biopsy under general anesthesia, establishment and decidualization of ESC with 10nM 17β-estradiol, 100nM progesterone and 0.5mM dibutyryl-cAMP (E/P/c), and manipulation of gap junctions in vitro via a combination of pharmacological or transgenic approaches. Main outcome measures: Decidualized ESC evaluated morphologically for epithelioid transformation, gap junctions by dye diffusion and Cx43, prolactin, VEGF and IL-6 expression by RT-PCR, Western and ELISA methods. Results: Cx43 accumulation and functional gap junctions between decidualized ESC increase concomitantly with morphological differentiation following E/P/c treatment. Disruption of gap junctions using pharmacological inhibitors or Cx43 shRNA prevents morphological differentiation and inhibits prolactin and VEGF secretion. By contrast, IL-6 secretion from decidualized ESC is augmented by both approaches. Conclusions: The findings suggest that decidualized ESC function as a coordinated secretory organ to regulate embryonic implantation via intercellular cooperation mediated by gap junctions. When adjacent cells can communicate through these junctions, decidual prolactin and VEGF secretion appears to be optimized for vascular development of the placental bed. Conversely, when intercellular communications are disrupted, angiogenesis is impaired and an inflammatory state is induced. [Copyright &y& Elsevier]

During implantation, the uterine stromal cells undergo terminal differentiation into decidual cells, which support the proper progression of maternal-embryo interactions to successful establishment of pregnancy. The decidual cells synthesize extracellular matrix (ECM) components, such as laminins and collagens, which assemble into a unique basal lamina-like network that surrounds these cells. The functional significance of this matrix during implantation is unknown. We previously showed that the transcription factor CCAAT enhancer-binding protein β (C/EBPβ) critically regulates decidualization in the mouse. We now provide evidence that C/EBPβ directly controls the Lamcl gene, which encodes a predominant laminin constituent of the ECM produced by the decidual cells. Suppression of Lamcl expression in mouse primary endometrial stromal cells prevented the assembly of this ECM and impaired stromal differentiation. Attenuation of expression of integrin β1, a major constituent of the integrin receptors targeted by decidual laminins, also inhibited this differentiation process. Disruption of laminin-integrin interactions led to impaired activation of the focal adhesion kinase, an integrin-mediated regulator of cytoskeletal remodeling during decidualization. To further analyze the role of the decidual ECM in modulating maternal-embryo interactions, we monitored trophoblast invasion into differentiating uterine stromal monolayers, using a co-culture system. Silencing of stromal Lamcl expression, which prevented formation of the basal lamina-like matrix, resulted in marked reduction in trophoblast outgrowth. Collectively, our findings identified C/EBPβ as a critical regulator of the unique ECM that controls decidual cell architecture and differentiation, and it provided new insights into the mechanisms by which the uterine stromal microenvironment controls the progression of embryo implantation. [ABSTRACT FROM AUTHOR]

A critical role of progesterone (P) during early pregnancy is to induce differentiation of the endometrial stromal cells into specialized decidual cells that support the development of the implanting embryo. The P-induced signaling pathways that participate in the formation and function of the decidual cells remain poorly understood. We report here that the expression of the bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is induced downstream of P action in the mouse uterine stroma during decidualization. To determine the function of BMP2 during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant mouse uterus undergo decidualization. When recombinant BMP2 was added to these stromal cultures, it markedly advanced the differentiation program. We also found that siRNA-mediated silencing of BMP2 expression in these cells efficiently blocked the differentiation process. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt4 expression by siRNAs greatly reduced stromal differentiation in vitro, indicating that it is a key mediator of BMP2-induced decidualization. We also observed a remarkable induction in the expression of BMP2 in human endometrial stromal cells during decidualization in vitro in response to steroids and cAMP. Addition of BMP2 to these cultures led to a robust enhancement of Wnt4 expression and stimulated the differentiation process. Collectively, our studies uncovered a unique conserved pathway involving BMP2 and Wnt4 that mediates P-induced stromal decidualization in the mouse and the human. [ABSTRACT FROM AUTHOR]

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR-retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR-RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR-RXR-corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR-RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR-RXR-corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex. [ABSTRACT FROM AUTHOR]

The 12/15-lipoxygenases (12/15-LOX) catalyze the stereo-specific oxygenation of arachidonic and linoleic acids into a complex series of signaling molecules, including the hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs). Our previous studies, using high density oligonucleotide microarrays, suggested a novel link between progesterone receptor (PR) signaling and 12/15-LOX-mediated fatty acid metabolism in preimplantation mouse uterus. In this paper, using PR knockout mice, we established that the transcripts encoding leukocyte-12/15-LOX (L-12/15-LOX) and epidermal-12/15-LOX (E-12/15-LOX) are indeed downstream targets of regulation by PR in the uterine surface epithelium. Maximal induction of both L- and E-12/15-LOX on the day of implantation resulted in a marked increase in the uterine levels of the eicosanoids, 12-HETE, 15-HETE, and 13-HODE. Mice with null mutation in L-12/15-LOX had significantly reduced uterine levels of arachidonic acid metabolites and exhibited a partial impairment in implantation. Complete blockade of uterine 12/15-LOX activity by a specific inhibitor led to greater than 80% reduction in a number of implantation sites relative to untreated controls. Cell-based assays indicated that 12-HETE, 15-HETE, and 13-HODE function as activating ligands of peroxisome proliferator-activated receptor γ (PPARγ), suggesting that this nuclear receptor could be a downstream target of 12/15LOX-derived metabolites in the preimplantation uterus. Consistent with this hypothesis, administration of rosiglitazone, a potent PPARγ-selective agonist, efficiently reversed inhibition of implantation by the 12/15-LOX-specific inhibitor. Rosiglitazone also induced a number of potential target genes of 12/15-LOX-derived metabolites in the pregnant uterus, indicating their regulation by PPARγ. Collectively, our results uncovered a novel signaling pathway in which progesterone-induced synthesis of the 12/15-LOX-derived lipid mediators activates PPARγ and its downstream gene networks, which in turn function as critical regulators of implantation in the mouse. [ABSTRACT FROM AUTHOR]