Decidualization dampens toll-like receptor mediated inflammatory responses in human endometrial stromal cells by upregulating IκBα.

Endometrial stromal cells (EnSCs) are the major cell type of the human endometrium and they undergo dramatic differentiation, termed decidualization, every month that enables them to be receptive to implantation. Appropriate decidualization and EnSC function is key for a successful pregnancy. EnSC function may be affected when the uterus is exposed to bacterial and viral infection. However, how human EnSCs respond to viral and bacterial components have not been well-studied and it remains unclear whether uterine innate immune responses change during decidualization. This study demonstrated that viral double-stranded RNA [Poly(I:C)] and bacterial lipopolysaccharide (LPS) upregulated undecidualized human EnSC production of a large array of proinflammatory cytokines and chemokines, and revealed that these immune responses were significantly dampened during decidualization in vitro and in vivo. This dampened response was associated with increased NFKBIA transcription during decidualization that leads to the accumulation of this negative regulator in decidualizing EnSCs that can bind to NFκB p65 and prevents its nuclear translocation and downstream Toll-like receptor signaling. These findings highlight that endometrial responses to infection may vary at different stages of the menstrual cycle which may be important for preparing the endometrium to support the growth of the semi-allogenic blastocyst. This work emphasizes the need to consider menstrual cycle stage, sex hormone levels and the differentiation status of cells when examining inflammatory responses in the future.

Antiphospholipid Antibodies Increase Endometrial Stromal Cell Decidualization, Senescence, and Inflammation via Toll-like Receptor 4, Reactive Oxygen Species, and p38 MAPK Signaling.

OBJECTIVE: Miscarriage affects 1 in 7 pregnancies, and antiphospholipid autoantibodies (aPLs) are one of the biggest risk factors for recurrent pregnancy loss. While aPLs target the endometrial stroma, little is known about their impact. Endometrial stromal cells (EnSCs) undergo decidualization each menstrual cycle, priming the uterus to receive implanting embryos. Thus, appropriate decidualization and EnSC function is key for establishment of a successful pregnancy. This study was undertaken to explore the effects of aPL on EnSC decidualization, senescence, and inflammation. METHODS: EnSCs under decidualizing conditions were exposed to aPL or control IgG alone or in the presence of either a Toll-like receptor 4 (TLR-4) antagonist, a p38 MAPK inhibitor, a reactive oxygen species (ROS) inhibitor, low molecular weight heparin (LMWH), or acetyl salicylic acid. Secretion of decidualization markers and inflammatory interleukin-8 were quantified by enzyme-linked immunosorbent assay, and senescence-associated ß-galactosidase activity was evaluated. In a mouse model of decidualization, aPL or control IgG was administered, and uterine expression levels of decidualization and inflammatory markers were quantified by real-time quantitative polymerase chain reaction. RESULTS: Antiphospholipid antibodies increased human EnSC decidualization, senescence, and inflammation. This phenotype was recapitulated in the mouse model. The decidualization and inflammatory responses were partially mediated by TLR-4 and p38 MAPK, while the decidualization and senescence responses were ROS-dependent. LMWH, commonly used to treat aPL-positive women at risk of obstetric complications, reduced the ability of aPL to increase EnSC decidualization and inflammation. CONCLUSION: These findings shed new light on the pathogenesis of pregnancy complications in women with aPLs and underscore the benefit of heparin in preventing pregnancy loss in this high-risk population.

TNF-α Regulated Endometrial Stroma Secretome Promotes Trophoblast Invasion.

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation.

Combinatorial actions of glucocorticoid and mineralocorticoid stress hormone receptors are required for preventing neurodegeneration of the mouse hippocampus.

Chronic stress contributes to numerous human pathologies including cognition impairments and psychiatric disorders. Glucocorticoids are primary stress hormones that activate two closely related nuclear receptors, the glucocorticoid (GR) and mineralocorticoid receptor (MR), that are both highly expressed in the hippocampus. To investigate potential combinatorial actions of hippocampal GR and MR, we developed mice with conditional knockout of both GR and MR in the hippocampus and compared them to their single knockout counterparts. Mice lacking MR alone or both GR and MR in the hippocampus exhibited altered expression of multiple CA2-specific neuronal markers and enhanced cue-dependent learning in a conditioned fear test. Provocatively, in contrast to the single knockouts, mice depleted of both GR and MR showed profound neurodegeneration of the hippocampus. Neuronal death was increased and neurogenesis was reduced in the dentate gyrus of the double knockout mice. Global gene expression assays of the knockout mice revealed a synergistic increase in the number of dysregulated genes in the hippocampus lacking both GR and MR. This large cohort of genes reliant on both GR and MR for expression was strongly associated with cell death and cell proliferation pathways. GR/MR complexes were detected in CA1 and dentate gyrus neurons suggesting receptor heterodimers contribute to the joint actions of GR and MR. These findings reveal an obligate role for MR signaling in regulating the molecular phenotype of CA2 neurons and demonstrate that combinatorial actions of GR and MR are essential for preserving dentate gyrus neurons and maintaining hippocampal health.

In Silico Exploration of the Potential Role of Acetaminophen and Pesticides in the Etiology of Autism Spectrum Disorder.

Recent epidemiological studies suggest that prenatal exposure to acetaminophen (APAP) is associated with increased risk of Autism Spectrum Disorder (ASD), a neurodevelopmental disorder affecting 1 in 59 children in the US. Maternal and prenatal exposure to pesticides from food and environmental sources have also been implicated to affect fetal neurodevelopment. However, the underlying mechanisms for ASD are so far unknown, likely with complex and multifactorial etiology. The aim of this study was to explore the potential effects of APAP and pesticide exposure on development with regards to the etiology of ASD by highlighting common genes and biological pathways. Genes associated with APAP, pesticides, and ASD through human research were retrieved from molecular and biomedical literature databases. The interaction network of overlapping genetic associations was subjected to network topology analysis and functional annotation of the resulting clusters. These genes were over-represented in pathways and biological processes (FDR p < 0.05) related to apoptosis, metabolism of reactive oxygen species (ROS), and carbohydrate metabolism. Since these three biological processes are frequently implicated in ASD, our findings support the hypothesis that cell death processes and specific metabolic pathways, both of which appear to be targeted by APAP and pesticide exposure, may be involved in the etiology of ASD. This novel exposures-gene-disease database mining might inspire future work on understanding the biological underpinnings of various ASD risk factors.

Deletion of hippocampal Glucocorticoid receptors unveils sex-biased microRNA expression and neuronal morphology alterations in mice.

Sex differences in the brain have prompted many researchers to investigate the underlying molecular actors, such as the glucocorticoid receptor (GR). This nuclear receptor controls gene expression, including microRNAs (miRNAs), in non-neuronal cells. Here, we investigated sex-biased effects of GR on hippocampal miRNA expression and neuronal morphology by generating a neuron-specific GR knockout mouse (Emx1-Nr3c1 -/-). The levels of 578 mature miRNAs were assessed using NanoString technology and, in contrast to males, female Emx1-Nr3c1 -/- mice showed a substantially higher number of differentially expressed miRNAs, confirming a sex-biased effect of GR ablation. Based on bioinformatic analyses we identified several transcription factors potentially involved in miRNA regulation. Functional enrichment analyses of the miRNA-mRNA interactions revealed pathways related to neuronal arborization and both spine morphology and density in both sexes. Two recognized regulators of dendritic morphology, CAMKII-α and GSK-3ß, increased their protein levels by GR ablation in female mice hippocampus, without changes in males. Additionally, sex-specific effects of GR deletion were observed on CA1 neuronal arborization and dendritic spine features. For instance, a reduced density of mushroom spines in apical dendrites was evidenced only in females, while a decreased length in basal dendrites was noted only in males. However, length and arborization of apical dendrites were reduced by GR ablation irrespective of the sex. Overall, our study provides new insights into the sex-biased GR actions, especially in terms of miRNAs expression and neuronal morphology in the hippocampus.

Yale School of Public Health Symposium: An overview of the challenges and opportunities associated with per- and polyfluoroalkyl substances (PFAS).

On December 13, 2019, the Yale School of Public Health hosted a symposium titled "Per- and Polyfluoroalkyl Substances (PFAS): Challenges and Opportunities" in New Haven, Connecticut. The meeting focused on the current state of the science on these chemicals, highlighted the challenges unique to PFAS, and explored promising opportunities for addressing them. It brought together participants from Yale University, the National Institute of Environmental Health Sciences, the University of Massachusetts Amherst, the University of Connecticut, the Connecticut Agricultural Experiment Station, the Connecticut Departments of Public Health and Energy and Environmental Protection, and the public and private sectors. Presentations during the symposium centered around several primary themes. The first reviewed the current state of the science on the health effects associated with PFAS exposure and noted key areas that warranted future research. As research in this field relies on specialized laboratory analyses, the second theme considered commercially available methods for PFAS analysis as well as several emerging analytical approaches that support health studies and facilitate the investigation of a broader range of PFAS. Since mitigation of PFAS exposure requires prevention and cleanup of contamination, the third theme highlighted new nanotechnology-enabled PFAS remediation technologies and explored the potential of green chemistry to develop safer alternatives to PFAS. The fourth theme covered collaborative efforts to assess the vulnerability of in-state private wells and small public water supplies to PFAS contamination by adjacent landfills, and the fifth focused on strategies that promote successful community engagement. This symposium supported a unique interdisciplinary coalition established during the development of Connecticut's PFAS Action Plan, and discussions occurring throughout the symposium revealed opportunities for collaborations among Connecticut scientists, state and local officials, and community advocates. In doing so, it bolstered the State of Connecticut's efforts to implement the ambitious initiatives that its PFAS Action Plan recommends.

Acetaminophen Attenuates invasion and alters the expression of extracellular matrix enzymes and vascular factors in human first trimester trophoblast cells.

Acetaminophen is one of the most common medications taken during pregnancy, considered safe for maternal health and fetal development. However, recent epidemiological studies have associated prenatal acetaminophen use with several developmental disorders in offspring. As acetaminophen can freely cross into and through the placenta, epidemiological associations with prenatal acetaminophen use may reflect direct actions on the fetus and/or the impact of altered placental functions. In the absence of rigorous mechanistic studies, our understanding of how prenatal acetaminophen exposure can cause long-term effects in offspring is limited. The objective of this study was to determine whether acetaminophen can alter key functions of a major placental cell type by utilizing immortalized human first trimester trophoblast cells. This study employed a comparative analysis with the nonsteroidal, anti-inflammatory drug aspirin, which has established effects in first trimester trophoblast cells. We report that immortalized trophoblast cells express the target proteins of acetaminophen and aspirin: cyclooxygenase (COX) -1 and -2. Unlike aspirin, acetaminophen significantly repressed the expression of angiogenesis and vascular remodeling genes in HTR-8/SVneo cells. Moreover, acetaminophen impaired trophoblast invasion by over 80%, while aspirin had no effect on invasion. Acetaminophen exposure reduced the expression of matrix metalloproteinase (MMP)-2 and -9 and increased the expression of tissue inhibitors of matrix metalloproteinases 2, leading to an imbalance in the ratio of proteolytic enzymes. Finally, a bioinformatic approach identified novel acetaminophen-responsive gene networks associated with key trophoblast functions and disease. Together these results suggest that prenatal acetaminophen use may interfere with critical trophoblast functions early in gestation, which may subsequently impact fetal development.

Updates on molecular and environmental determinants of luteal progesterone production.

Progesterone, a critical hormone in reproduction, is a key sex steroid in the establishment and maintenance of early pregnancy and serves as an intermediary for synthesis of other steroid hormones. Progesterone production from the corpus luteum is a tightly regulated process which is stimulated and maintained by multiple factors, both systemic and local. Multiple regulatory systems, including classic mediators of gonadotropin stimulation such as the cAMP/PKA pathway and TGFß-mediated signaling pathways, as well as local production of hormonal factors, exist to promote granulosa cell function and physiological fine-tuning of progesterone levels. In this manuscript, we provide an updated narrative review of the known mediators of human luteal progesterone and highlight new observations regarding this important process, focusing on studies published within the last five years. We will also review recent evidence suggesting that this complex system of progesterone production is sensitive to disruption by exogenous environmental chemicals that can mimic or interfere with the activities of endogenous hormones.

Gene X environment: the cellular environment governs the transcriptional response to environmental chemicals.

BACKGROUND: An individual's response to environmental exposures varies depending on their genotype, which has been termed the gene-environment interaction. The phenotype of cell exposed can also be a key determinant in the response to physiological cues, indicating that a cell-gene-environment interaction may exist. We investigated whether the cellular environment could alter the transcriptional response to environmental chemicals. Publicly available gene expression array data permitted a targeted comparison of the transcriptional response to a unique subclass of environmental chemicals that alter the activity of the estrogen receptor, xenoestrogens. RESULTS: Thirty xenoestrogens were included in the analysis, for which 426 human gene expression studies were identified. Comparisons were made for studies that met the predefined criteria for exposure length, concentration, and experimental replicates. The cellular response to the phytoestrogen genistein resulted in remarkably unique transcriptional profiles in breast, liver, and uterine cell-types. Analysis of gene regulatory networks and molecular pathways revealed that the cellular context mediated the activation or repression of functions important to cellular organization and survival, including opposing effects by genistein in breast vs. liver and uterine cell-types. When controlling for cell-type, xenoestrogens regulate unique gene networks and biological functions, despite belonging to the same class of environmental chemicals. Interestingly, the genetic sex of the cell-type also strongly influenced the transcriptional response to xenoestrogens in the liver, with only 22% of the genes significantly regulated by genistein common between male and female cells. CONCLUSIONS: Our results demonstrate that the transcriptional response to environmental chemicals depends on a variety of factors, including the cellular context, the genetic sex of a cell, and the individual chemical. These findings highlight the importance of evaluating the impact of exposure across cell-types, as the effect is responsive to the cellular environment. These comparative genetic results support the concept of a cell-gene-environment interaction.

The Selective Progesterone Receptor Modulator Ulipristal Acetate Inhibits the Activity of the Glucocorticoid Receptor.

CONTEXT: The selective progesterone modulator ulipristal acetate (ulipristal) offers a much-needed therapeutic option for the clinical management of uterine fibroids. Although ulipristal initially passed safety evaluations in Europe, postmarketing analysis identified cases of hepatic injury and failure, leading to restrictions on the long-term use of ulipristal. One of the factors potentially contributing to significant side effects with the selective progesterone modulators is cross-reactivity with other steroid receptors. OBJECTIVE: To determine whether ulipristal can alter the activity of the endogenous glucocorticoid receptor (GR) in relevant cell types. DESIGN: Immortalized human uterine fibroid cells (UtLM) and hepatocytes (HepG2) were treated with the synthetic glucocorticoid dexamethasone and/or ulipristal. Primary uterine fibroid tissue was isolated from patients undergoing elective gynecological surgery and treated ex vivo with dexamethasone and/or ulipristal. In vivo ulipristal exposure was performed in C57Bl/6 mice to measure the effect on basal gene expression in target tissues throughout the body. RESULTS: Dexamethasone induced the expression of established glucocorticoid-target genes period 1 (PER1), FK506 binding protein 51 (FKBP5), and glucocorticoid-induced leucine zipper (GILZ) in UtLM and HepG2 cells, whereas cotreatment with ulipristal blocked the transcriptional response to glucocorticoids in a dose-dependent manner. Ulipristal inhibited glucocorticoid-mediated phosphorylation, nuclear translocation, and DNA interactions of GR. Glucocorticoid stimulation of PER1, FKBP5, and GILZ was abolished by cotreatment with ulipristal in primary uterine fibroid tissue. The expression of glucocorticoid-responsive genes was decreased in the lung, liver, and uterus of mice exposed to 2 mg/kg ulipristal. Interestingly, transcript levels of Fkbp5 and Gilz were increased in the hippocampus and pituitary. CONCLUSIONS: These studies demonstrate that ulipristal inhibits endogenous glucocorticoid signaling in human fibroid and liver cells, which is an important consideration for its use as a long-term therapeutic agent.

Determining the endocrine disruption potential of industrial chemicals using an integrative approach: Public databases, in vitro exposure, and modeling receptor interactions.

Environmental and occupational exposure to industrial chemicals has been linked to toxic and carcinogenic effects in animal models and human studies. However, current toxicology testing does not thoroughly explore the endocrine disrupting effects of industrial chemicals, which may have low dose effects not predicted when determining the limit of toxicity. The objective of this study was to evaluate the endocrine disrupting potential of a broad range of chemicals used in the petrochemical sector. Therefore, 139 chemicals were classified for reproductive toxicity based on the United Nations Globally Harmonized System for hazard classification. These chemicals were evaluated in PubMed for reported endocrine disrupting activity, and their endocrine disrupting potential was estimated by identifying chemicals with active nuclear receptor endpoints publicly available databases. Evaluation of ToxCast data suggested that these chemicals preferentially alter the activity of the estrogen receptor (ER). Four chemicals were prioritized for in vitro testing using the ER-positive, immortalized human uterine Ishikawa cell line and a range of concentrations below the reported limit of toxicity in humans. We found that 2,6-di-tert-butyl-p-cresol (BHT) and diethanolamine (DEA) repressed the basal expression of estrogen-responsive genes PGR, NPPC, and GREB1 in Ishikawa cells, while tetrachloroethylene (PCE) and 2,2'-methyliminodiethanol (MDEA) induced the expression of these genes. Furthermore, low-dose combinations of PCE and MDEA produced additive effects. All four chemicals interfered with estradiol-mediated induction of PGR, NPPC, and GREB1. Molecular docking demonstrated that these chemicals could bind to the ligand binding site of ERα, suggesting the potential for direct stimulatory or inhibitory effects. We found that these chemicals altered rates of proliferation and regulated the expression of cell proliferation associated genes. These findings demonstrate previously unappreciated endocrine disrupting effects and underscore the importance of testing the endocrine disrupting potential of chemicals in the future to better understand their potential to impact public health.

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

New models of lipopolysaccharide-induced implantation loss reveal insights into the inflammatory response.

PROBLEM: Chronic endometritis, inflammation of the uterizzvvne lining caused by common gram-negative bacterial strains or mycoplasma, has been associated with unexplained implantation failure and infertility. However, limited models of bacteria-induced implantation loss exist to study the molecular changes that occur in vivo. The goal of this study was to provide a new resource to study the process of bacteria-induced inflammation and implantation loss utilizing common experimental models: C57Bl/6 mice and primary human endometrial stromal cells. METHOD OF STUDY: Prior to implantation, mated C57Bl/6 females were administered vehicle (saline) or gram-negative bacterial lipopolysaccharide (LPS) at a range of concentrations by intraperitoneal injection. Implantation sites were counted, and uteri were harvested to evaluate the molecular changes that accompany LPS-mediated implantation loss. Primary human endometrial stromal cells were decidualized in vitro in the presence and absence of LPS. Total RNA and conditioned media were harvested to evaluate the expression of known decidualization-associated genes and various cytokines and chemokines. RESULTS: Lipopolysaccharide treatment resulted in fewer implantation sites in mice, decreased expression of decidualization-associated genes, and altered expression and release of cytokines and chemokines. Immunohistological analysis of the uterus from LPS-exposed mice demonstrated increased apoptosis and decreased proliferation during decidualization. CONCLUSION: Lipopolysaccharide exposure disrupted implantation and decidualization in mice and human endometrial stromal cells. This model could be used to study the pathophysiology of implantation failure in patients with chronic endometritis or to test potential therapeutic interventions.

Glucocorticoid signaling regulates cell invasion and migration in the human first-trimester trophoblast cell line Sw.71.

PROBLEM: The development of the placenta and its functions are sensitive to infection and stress, which can activate the hypothalamic-pituitary-adrenal axis. Adrenally produced glucocorticoids are the body's primary mediators of the inflammatory and stress response. Although the glucocorticoid receptor (GR) is expressed in all human villous trophoblast tissue, the effect of glucocorticoids on placentation is not well understood. METHOD OF STUDY: Using microarray analysis, we identified the glucocorticoid-regulated transcriptional profile in the immortalized first-trimester extravillous trophoblast cell line Swan.71 (Sw.71). RESULTS: The synthetic glucocorticoid dexamethasone significantly regulated 3829 genes, including genes associated with cell movement, growth, and survival. SERPINE1, an inhibitor of trophoblast invasion, was induced by glucocorticoids in Sw.71 cells and is associated with the pathogenesis of preeclampsia. Glucocorticoid treatment induced recruitment of activated polymerase II and GR to the SERPINE1 promoter, suggesting a mechanism for transcriptional regulation. Functionally, glucocorticoid treatment inhibited cell proliferation, migration, and invasion. CONCLUSION: These findings suggest that glucocorticoids regulate extravillous trophoblast functions by altering the gene expression profile, which may contribute to the pathogenesis of reproductive disorders such as preeclampsia and IUGR.

Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus.

BACKGROUND: Female reproductive tract development is sensitive to the endocrine-disrupting potential of environmental estrogens. Early-life exposure to the dietary phytoestrogen genistein impairs fertility and persistently alters the transcriptome in the oviduct and uterus of rodents. Glucocorticoid signaling, which has recently been shown to be essential for normal fertility in the female mouse uterus, is antagonized by genistein. OBJECTIVE: Our goal was to determine whether early-life exposure to genistein disrupts glucocorticoid signaling in the mouse uterus, which may contribute to infertility. METHODS: Female C57Bl/6 mice were exposed to either 50 mg/kg per day genistein, 10 µg/kg per day estradiol, or vehicle (corn oil) on postnatal days 1-5 (PND1-5), and then treated with the synthetic glucocorticoid dexamethasone (Dex: 1 mg/kg) or vehicle (saline) on PND5, at weaning on PND21, or as adults on PND56 following adrenalectomy and ovariectomy to evaluate glucocorticoid responsiveness. Uteri were isolated following treatment for gene expression or chromatin immunoprecipitation. RESULTS: Neonatal exposure to genistein altered the uterine transcriptome of adult mice and caused substantial changes to the transcriptional response to glucocorticoids. Although expression of the glucocorticoid receptor was not affected, genistein exposure disrupted glucocorticoid receptor recruitment to specific regulatory sites in target genes. Many genes involved in chromatin remodeling were dysregulated in genistein-exposed mice, suggesting that epigenetic reprograming may contribute to the altered glucocorticoid response of the uterus following early-life exposure to genistein. These changes affected the biological activity of glucocorticoids within the uterus, as glucocorticoids antagonized the proliferative effects of estradiol in the uterus of control mice but not genistein-exposed mice. CONCLUSIONS: Our findings suggest that disruption of glucocorticoid signaling due to early-life exposure to environmental estrogens may in part render the uterus unable to support implantation. https://doi.org/10.1289/EHP1575.

Glucocorticoid Signaling in Health and Disease: Insights From Tissue-Specific GR Knockout Mice.

Glucocorticoids are adrenally produced hormones critically involved in development, general physiology, and control of inflammation. Since their discovery, glucocorticoids have been widely used to treat a variety of inflammatory conditions. However, high doses or prolonged use leads to a number of side effects throughout the body, which preclude their clinical utility. The primary actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a transcription factor that regulates many complex signaling pathways. Although GR is nearly ubiquitous throughout the body, glucocorticoids exhibit cell- and tissue-specific effects. For example, glucocorticoids stimulate glucose production in the liver, reduce glucose uptake in the skeletal muscle, and decrease insulin secretion from the pancreatic ß-cells. Mouse models represent an important approach to understanding the dynamic functions of GR signaling in normal physiology, disease, and resistance. In the absence of a viable GR null model, gene-targeting techniques utilizing promoter-driven recombination have provided an opportunity to characterize the tissue-specific actions of GR. The aim of the present review is to describe the organ systems in which GR has been conditionally deleted and summarize the functions ascribed to glucocorticoid action in those tissues.

Stress and the HPA Axis: Balancing Homeostasis and Fertility.

An organism's reproductive fitness is sensitive to the environment, integrating cues of resource availability, ecological factors, and hazards within its habitat. Events that challenge the environment of an organism activate the central stress response system, which is primarily mediated by the hypothalamic-pituitary-adrenal (HPA) axis. The regulatory functions of the HPA axis govern the cardiovascular and metabolic system, immune functions, behavior, and reproduction. Activation of the HPA axis by various stressors primarily inhibits reproductive function and is able to alter fetal development, imparting a biological record of stress experienced in utero. Clinical studies and experimental data indicate that stress signaling can mediate these effects through direct actions in the brain, gonads, and embryonic tissues. This review focuses on the mechanisms by which stress activation of the HPA axis impacts fertility and fetal development.

Pioneer Factors FOXA1 and FOXA2 Assist Selective Glucocorticoid Receptor Signaling in Human Endometrial Cells.

Successful pregnancy relies on dynamic control of cell signaling to achieve uterine receptivity and the necessary biological changes required for endometrial decidualization, embryo implantation, and fetal development. Glucocorticoids are master regulators of intracellular signaling and can directly regulate embryo implantation and endometrial remodeling during murine pregnancy. In immortalized human uterine cells, we have shown that glucocorticoids and estradiol (E2) coregulate thousands of genes. Recently, glucocorticoids and E2 were shown to coregulate the expression of Left-right determination factor 1 (LEFTY1), previously implicated in the regulation of decidualization. To elucidate the molecular mechanism by which glucocorticoids and E2 regulate the expression of LEFTY1, immortalized and primary human endometrial cells were evaluated for gene expression and receptor recruitment to regulatory regions of the LEFTY1 gene. Glucocorticoid administration induced expression of LEFTY1 messenger RNA and protein and recruitment of the glucocorticoid receptor (GR) and activated polymerase 2 to the promoter of LEFTY1. Glucocorticoid-mediated recruitment of GR was dependent on pioneer factors FOXA1 and FOXA2. E2 was found to antagonize glucocorticoid-mediated induction of LEFTY1 by reducing recruitment of GR, FOXA1, FOXA2, and activated polymerase 2 to the LEFTY1 promoter. Gene expression analysis identified several genes whose glucocorticoid-dependent induction required FOXA1 and FOXA2 in endometrial cells. These results suggest a molecular mechanism by which E2 antagonizes GR-dependent induction of specific genes by preventing the recruitment of the pioneer factors FOXA1 and FOXA2 in a physiologically relevant model.

Generating diversity in human glucocorticoid signaling through a racially diverse polymorphism in the beta isoform of the glucocorticoid receptor.

Alternative splicing of the human glucocorticoid receptor gene generates two isoforms, hGRα and hGRß. hGRß functions as a dominant-negative regulator of hGRα activity and but also has inherent transcriptional activity, collectively altering glucocorticoid sensitivity. Single-nucleotide polymorphisms in the 3' UTR of hGRß have been associated with altered receptor protein expression, glucocorticoid sensitivity, and disease risk. Characterization of the hGRß G3134T polymorphism has been limited to a relatively small, homogenous population. The objective of this study was to determine the prevalence of hGRß G3134T in a diverse population and assess the association of hGRß G3134T in this population with physiological outcomes. In a prospective cohort study, 3730 genetically diverse participants were genotyped for hGRß G3134T and four common GR polymorphisms. A subset of these participants was evaluated for clinical and biochemical measurements. Immortalized human osteosarcoma cells (U-2 OS), stably transfected with wild-type or G3134T hGRß, were evaluated for receptor expression, stability, and genome-wide gene expression. Glucocorticoid-mediated gene expression profiles were investigated in primary macrophages isolated from participants. In a racially diverse population, the minor allele frequency was 74% (50.7% heterozygous carriers and 23.3% homozygous minor allele), with a higher prevalence in Caucasian non-Hispanic participants. After adjusting for confounding variable, carriers of hGRß G3134T were more likely to self-report allergies, have higher serum cortisol levels, and reduced cortisol suppression in response to low-dose dexamethasone. The presence of hGRß G3134T in U-2 OS cells increased hGR mRNA stability and protein expression. Microarray analysis revealed that the presence of the hGRß G3134T polymorphism uniquely altered gene expression profiles in U-2 OS cells and primary macrophages. hGRß G3134T is significantly present in the study population and associated with race, self-reported disease, and serum levels of glucocorticoids. Underlying these health differences may be changes in gene expression driven by altered receptor stability.