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Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption.

Glucocorticoids (GC) are crucial regulators of homeostasis and function. Despite its negative side effects, glucocorticoid therapy in neonates is widely used antenatally for accelerating fetal lung maturation in cases of preterm birth. GC action is mediated via glucocorticoid receptors — ligand-activated transcription factors. Cell death and viability in the neonatal brain are regulated by many factors, but the glucocorticoid receptor signalling is high above them. The present work studies the changes in the expression of genes for apoptosis regulators with Bcl-2 homology (BH) domains (Bcl-xL, Bax, Bim, Bok, Bid) in the neonatal rat hippocampus after dexamethasone (DEX) and CpdA administration. CpdA is a dissociative ligand — glucocorticoid receptor modulator — that shifts glucocorticoid receptor (GR) activity toward transrepression. Ligands administration to P2 pups caused different patterns of timeline changes in the expression of the studied genes. We observed the first increase in the mRNA level of the genes which have glucocorticoid response element (GRE) (Bcl-xL, Bim) in their promoter 30 min after DEX administration. Activated GR action on cells in the neonatal hippocampus is complex and long-lasting; it could also contain receptor homo- and hetero-dimerisation. Using rat pheochromocytoma PC12 cells as a test system, we assessed GR-GR and GR-MR (mineralocorticoid receptor) dimerisation with proximity ligation assay (PLA) assay separately in the nucleus and cytoplasm after DEX and CpdA administration. An increase in GR-GR dimers in the cell nucleus was observed only after DEX administration. In the cell cytoplasm, we observed a gradual (DEX more than CpdA) increase in the number of both GR-GR and GR-MR dimers.

As a natural dietary ingredient, berberine possesses multiple biological activities including anti-inflammatory effects. In this work, glucocorticoid receptor (GR)-mediated alleviation of inflammation by berberine was investigated by a combination of in vitro, in silico, and in vivo approaches. The fluorescence polarization assay showed that berberine bound to GR with an IC50 value of 9.14 ± 0.16 pM. Molecular docking and molecular dynamics simulation suggested that berberine bound stably to the active site of GR via hydrogen bonding and hydrophobic interactions. Berberine induced GR nuclear translocation but did not activate the glucocorticoid response element in HeLa cells. Furthermore, both gene and protein expressions of PEPCK were significantly attenuated by berberine in HepG2 cells. Interestingly, berberine downregulated CBG mRNA and protein levels without up-regulating TAT mRNA and protein levels in HepG2 cells, demonstrating its dissociated characteristics that could separate transrepression from transactivation. In addition, the in vitro and in vivo anti-inflammatory effects of berberine were confirmed in lipopolysaccharide-induced RAW 264.7 cells and in a mouse model of allergic contact dermatitis, respectively. In conclusion, berberine might serve as a potential selective GR modulator.

Background GAS5 contains a hormone response element that can induce cell apoptosis in breast cancer. It is known that cell apoptosis and hormone response play crucial roles in polycystic ovary syndrome (PCOS), indicating the potential involvement of GAS5 in PCOS. This study was performed to investigate the potential involvement of GAS5 and IL-6 (a critical player in PCOS) in PCOS. Methods Research subjects of this study included 60 PCOS patients and 60 healthy controls. The expression levels of GAS5 and IL-6 in plasma of both patients and controls were measured by qPCR and ELISA, respectively. Cell transfections were performed to analyze the interaction between GAS5 and IL-6. Cell apoptosis was analyzed by cell apoptosis assay. Results GAS5 was upregulated in plasma of PCOS patients. The expression levels of GAS5 were positively correlated with the expression levels of IL-6. Altered expression levels of GAS5 and IL-6 distinguished PCOS patients from healthy controls. In cells of a granulosa-like tumor cell line (KGN), overexpression of GAS5 led to upregulated IL-6, while silencing of GAS5 played an opposite role. Cell apoptosis analysis showed that overexpression of GAS5 significantly decreased apoptosis rate of KGN cells. Silencing of GAS5 increased the rate of KGN cell apoptosis. Conclusions GAS5 is upregulated in PCOS and regulates cell apoptosis and the expression of IL-6.

SIRT2 is a NAD+‐dependent deacetylase that deacetylates a diverse array of protein substrates and is involved in many cellular processes, including regulation of inflammation. However, its precise role in the inflammatory process has not completely been elucidated. Here, we identify heat‐shock protein 90α (Hsp90α) as novel substrate of SIRT2. Functional investigation suggests that Hsp90 is deacetylated by SIRT2, such that overexpression and knock‐down of SIRT2 altered the acetylation level of Hsp90. This subsequently resulted in disassociation of Hsp90 with glucocorticoid receptor (GR), and translocation of GR to the nucleus. This observation was further confirmed by glucocorticoid response element (GRE)‐driven reporter assay. Nuclear translocation of GR induced by SIRT2 overexpression repressed the expression of inflammatory cytokines, which were even more prominent under lipopolysaccharide (LPS) stimulation. Conversely, SIRT2 knock‐down resulted in the up‐regulation of cytokine expression. Mutation analysis indicated that deacetylation of Hsp90 at K294 is critical for SIRT2‐mediated regulation of cytokine expression. These data suggest that SIRT2 reduces the extent of LPS‐induced inflammation by suppressing the expression of inflammatory factors via SIRT2‐Hsp90‐GR axis.

While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC(50) values of 15.30 μM and 19.26 μM respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein’s activity in OVCAR5 cells, confirming baicalein’s activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4 weeks at a dose of 25 mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.

This review addresses the molecular mechanisms of corticotropin-releasing factor (CRF) regulation in the hypothalamus under stress and stress resilience. CRF in the hypothalamus plays a central role in regulating the stress response. CRF stimulates adrenocorticotropic hormone (ACTH) release from the anterior pituitary. ACTH stimulates glucocorticoid secretion from the adrenal glands. Glucocorticoids are essential for stress coping, stress resilience, and homeostasis. The activated hypothalamic-pituitary-adrenal axis is suppressed by the negative feedback from glucocorticoids. Glucocorticoid-dependent repression of cAMP-stimulated Crf promoter activity is mediated by both the negative glucocorticoid response element and the serum response element. Conversely, the inducible cAMP-early repressor can suppress the stress response via inhibition of the cAMP-dependent Crf gene, as can the suppressor of cytokine signaling-3 in the hypothalamus. CRF receptor type 1 is mainly involved in a stress response, depression, anorexia, and seizure, while CRF receptor type 2 mediates “stress coping” mechanisms such as anxiolysis in the brain. Differential effects of FK506-binding immunophilins, FKBP4 and FKBP5, contribute to the efficiency of glucocorticoids under stress resilience. Together, a variety of factors contribute to stress resilience. All these factors would have the differential roles under stress resilience.

Women are vulnerable to developing mental disorders that are associated with circulating estrogens. Estrogens, especially 17β-estradiol (E2), have a wide array of effects on the brain, affecting many behavioral endpoints associated with mental illness. By using a total estrogen receptor (ER) α knockout (KO), an ERα knock in/knock out (KIKO) that lacks a functional DNA-binding domain, and wild type (WT) controls treated with either oil or E2, we evaluated ERα signaling, dependent and independent of the estrogen response element (ERE), on avoidance behavior, social interactions and memory, and palatable ingestive behavior using the open field test, the elevated plus maze, the light dark box, the 3-chamber test, and palatable feeding. We found that ERα does not mediate control of anxiety-like behaviors but rather yielded differences in locomotor activity. In evaluating social preference and social recognition memory, we observed that E2 may modulate these measures in KIKO females but not KO females, suggesting that ERE-independent signaling is likely involved in sociability. Lastly, observations of palatable (high-fat) food intake suggested an increase in palatable eating behavior in oil-treated KIKO females. Oil-treated KO females had a longer latency to food intake, indicative of an anhedonic phenotype compared to oil-treated WT and KIKO females. We have observed that social-related behaviors are potentially influenced by ERE-independent ERα signaling and hedonic food intake requires signaling of ERα.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LβT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

Inflammation can deregulate the testicular functions of steroidogenesis and spermatogenesis, consequently contributing to male infertility. Animals and cells treated with lipopolysaccharide (LPS) exhibit infection- and inflammation-induced testicular dysfunction. However, the precise mechanisms affecting steroidogenesis and spermatogenesis in response to LPS-treatment remain poorly understood. We isolated distinct testicular cells including spermatocytes, round spermatids and late spermatids to analyze distribution of peroxisome proliferator-activated receptor (PPAR) family, plays central roles in the regulation of metabolism. Our results suggested Pparα/Pparγ mRNA was highly expressed in late spermatids, while Pparβ mRNA was highly expressed in round spermatids. To analyze the effect of LPS on testicular cells, we established an LPS infection model using primary Sertoli cells and testicular cell lines (TM4, GC2 and MLTC1). We observed that PPARγ and SIRT1 were concentrated in the nuclear region and that the mRNA expression levels of antioxidative enzymes (Cat and Homx1) and PPARγ were upregulated in primary Sertoli cells after LPS-treatment. Moreover, luciferase reporter gene assays of the testicular cell lines revealed that the activity of the PPAR response element (PPRE) was significantly increased. Importantly, the transcriptional activity of the androgen response element was significantly reduced, whereas activity of estrogen response element was strongly induced in LPS-treated TM4 cells, consistent with the RT-PCR results. Meanwhile, the qRT-PCR results revealed that the LPS-induced upregulation of Ar mRNA in MLTC1 cells and Erβ mRNA in TM4 cells were significantly recovered after treatment with the specific PPARγ-antagonist GW9662. In addition, we also found that LPS induced alterations in enzymes involved in steroidogenesis in testicular cell lines. Taken together, our results revealed that LPS may induce PPAR transcriptional activity to disturb estrogen/androgen receptor expression and impair steroidogenesis and ROS metabolism in testicular cells.

Objective The endogenous estrogen 17β-estradiol (E2) promotes metabolic homeostasis in premenopausal women. In a mouse model of post-menopausal metabolic syndrome, we reported that estrogens increased energy expenditure, thus preventing estrogen deficiency-induced adiposity. Estrogens' prevention of fat accumulation was associated with increased serum concentrations of fibroblast growth factor 21 (FGF21), suggesting that FGF21 participates in estrogens' promotion of energy expenditure. Methods We studied the effect of E2 on FGF21 production and the role of FGF21 in E2 stimulation of energy expenditure and prevention of adiposity, using female estrogen receptor (ER)- and FGF21-deficient mice fed a normal chow and a cohort of ovariectomized women from the French E3N prospective cohort study. Results E2 acting on the hepatocyte ERα increases hepatic expression and production of FGF21 in female mice. In vivo activation of ERα increases the transcription of Fgf21 via an estrogen response element outside the promoter of Fgf21. Treatment with E2 increases oxygen consumption and energy expenditure and prevents whole body fat accumulation in ovariectomized female WT mice. The effect of E2 on energy expenditure is not observed in FGF21-deficient mice. While E2 treatment still prevents fat accumulation in FGF21-deficient mice, this effect is decreased compared to WT mice. In an observational cohort of ovariectomized women, E2 treatment was associated with lower serum FGF21 concentrations, which may reflect a healthier metabolic profile. Conclusions In female mice, E2 action on the hepatocyte ERα increases Fgf21 transcription and FGF21 production, thus promoting energy expenditure and partially decreasing fat accumulation., Highlights • Activation of the hepatocyte ERα increases FGF21 production in female mice. • ERα increases Fgf21 transcription in vivo via an estrogen response element. • Estrogen stimulation of energy expenditure is lost in female FGF21-deficient mice. • In women, estrogen treatment is associated with lower serum FGF21 concentrations.

Breast cancer is the most common malignancy in women worldwide. Estrogen receptor α (ERα) is expressed in ∼70% of breast cancer cases and promotes estrogen-dependent cancer progression. In the present study, we identified OTU domain-containing 7B (OTUD7B), a deubiquitylase belonging to A20 subgroup of ovarian tumor protein superfamily, as a bona fide deubiquitylase of ERα in breast cancer. OTUD7B expression was found to be positively correlated with ERα in breast cancer and associated with poor prognosis. OTUD7B could interact with, deubiquitylate, and stabilize ERα in a deubiquitylation activity-dependent manner. Depletion of OTUD7B decreased ERα protein level, the expression of ERα target genes, and the activity of estrogen response element in breast cancer cells. In addition, OTUD7B depletion significantly decreased ERα-positive breast cancer cell proliferation and migration. Finally, overexpression of ERα could rescue the suppressive effect induced by OTUD7B depletion, suggesting that the ERα status was essential to the function of OTUD7B in breast carcinogenesis. In conclusion, our study revealed an interesting post-translational mechanism between ERα and OTUD7B in ERα-positive breast cancer. Targeting the OTUD7B–ERα complex may prove to be a potential approach to treat patients with ERα-positive breast cancer.

The glucocorticoid receptor (GR) is a nuclear hormone receptor critical to the regulation of energy metabolism and the inflammatory response. The actions of GR are highly dependent on cell type and environmental context. Here, we demonstrate the necessity for liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver-specificity of GR action. In normal mouse liver, the HNF4 motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites found within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodelled, with both loss and gain of GR recruitment evident. Lost sites are characterised by HNF4 motifs and weak GRE motifs. Gained sites are characterised by strong GRE motifs, and typically show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is further demonstrated by evidence of an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

Previously, we had established a highly sensitive trap vector system for the efficient isolation of reporter cells for a certain condition of interest. In this study, we used this system to screen reporter cells that express the luciferase and enhanced green fluorescent protein genes in response to dexamethasone, a glucocorticoid receptor agonist to facilitate glucocorticoid signaling research. In total, 10 clones were isolated. The insertion sites of the trap vector were analyzed using 5' rapid amplification of cDNA ends (5' RACE), whereupon LPIN1, PKP2, and FKBP5 were identified as genes that were upregulated by the dexamethasone treatment. Specifically, PKP2 has not previously been focused as a gene that responds to glucocorticoids. The PKP2 mRNA was analyzed and induction of the endogenous gene was confirmed by real-time polymerase chain reaction. Given that PKP2 does not appear to have a consensus glucocorticoid response element (GRE) sequence, this reporter clone could supplement the current GRE-based reporter systems that are prevalently used. Because different clones showed different responses to glucocorticoids, these clones should provide more information than analysis with a single reporter clone. This paper demonstrates that the previously developed trap vector technology can contribute to the rapid construction of drug evaluation systems.

Estrogens and estrogenic endocrine disrupting chemicals can cause gonadal feminization in some vertebrates mainly through estrogen receptor (ER), but the underlying molecular mechanisms are unclear. The present study aimed to identify ER target genes involved in estrogen-caused gonadal feminization in Xenopus laevis. Based on our recent transcriptomic data that 10 nM 17β-estradiol (E2) altered gene transcription in feminizing gonads of male X. laevis at NF stages 48, 50, and 52, we searched estrogen response element (ERE) using the Dragon ERE Finder software in the promoter region of all the E2-regulated genes. As a result, 163 genes containing ERE sequence were identified as predicted ER target genes at NF stage 50 (on the 14th day postfertilization), a crucial stage for gonadal feminization. Then, some of these predicted ER target genes were further investigated, mainly including the genes that were suggested to be involved in E2-caused gonadal feminization and genes being dramatically up or down-regulated by E2. Fifteen genes were demonstrated to be responsive to E2, in turn ER antagonist blocked the E2-regulated transcription. Finally, we identified 10 genes that can bind to ERα by a chromatin immunoprecipitation-qPCR. Taken together, we identified the 10 genes that contain predicted ERE sequences, are responsive to estrogen and ER antagonist, and have ability to bind to ER as ER target genes, including pglyrp2, apoa1, fgb, tdo2, ca6, nags, cpb2, tmprss6, nudc, zwilch. Our results could help to improve the understanding of the molecular mechanisms for gonadal feminization caused by estrogenic endocrine disrupting chemicals in X. laevis, and even in other species.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Glucocorticoids are potent anti-inflammatory drugs that are used to treat an extraordinary range of human disease, including COVID-19, underscoring the ongoing importance of understanding their molecular mechanisms. Early studies of GR signaling led to broad acceptance of models in which glucocorticoid receptor (GR) monomers tether repressively to inflammatory transcription factors, thus abrogating inflammatory gene expression. However, newer data challenge this core concept and present an exciting opportunity to reframe our understanding of GR signaling. Here, we present an alternate, two-part model for transcriptional repression by glucocorticoids. First, widespread GR-mediated induction of transcription results in rapid, primary repression of inflammatory gene transcription and associated enhancers through competition-based mechanisms. Second, a subset of GR-induced genes, including targets that are regulated in coordination with inflammatory transcription factors such as NF-κB, exerts secondary repressive effects on inflammatory gene expression. Within this framework, emerging data indicate that the gene set regulated through the cooperative convergence of GR and NF-κB signaling is central to the broad clinical effectiveness of glucocorticoids in terminating inflammation and promoting tissue repair.

Background Thyroid hormone participates in lipid metabolism regulation. However, the effects on triacyleride or triacylglycerol metabolism are complex and not fully clarified yet. In this study, we try to identify novel thyroid hormone-targeting lipogenic metabolic genes and analyze their molecular regulative mechanism. Method Thirty-five promoters of twenty-nine human lipogenic regulative enzyme genes were constructed into pXP1 luciferase reporter plasmid (PFK2/FBP2-luc) and transfected into HeGP2 cells, respectively. Gene expression induced by triiodothyronine (T3) was detected by luciferase assay. The T3-activated gene promoter was then analyzed by sequence analysis, deletion and mutation, and electrophoretic mobility shift assay (EMSA). Results After 10 nM T3 stimulation for 36 h, phosphogluconate dehydrogenase, malic enzyme, Glycerol-3-phosphate acyltransferase (GPAT) 3, and 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 were significantly activated, respectively. A AGGTCA-like-direct-repeat-4 consensus thyroid hormone response element (DR4-TRE)-like sequence was found in the GPAT3 promoter, which was then verified to be necessary for T3-induced GPAT3 activation by gene deletion and mutation analysis. EMSA further identified that T3-thyroid receptor (TR) α-retinoid-X receptor (RXR) complex directly bound on the GPAT3 promoter. Conclusion Triiodothyronine could activate the GPAT3 through DR4-TRE-like sequence binding to participate in lipogenic regulation. AGPAT2 may be another thyroid hormone target enzyme.

Background We previously identified PIWIL1 as an oncogene involved in endometrial carcinogenesis. However, the mechanism of Piwil1 mediated regulation of tumorigenesis remains poorly understood. Methods The expression levels of target genes in endometrial cancer cells were detected by quantitative reverse transcription-PCR (RT-qPCR) and western blotting. Up- or down-regulation of ERα or PIWIL1 was achieved by transient transfection with expressing plasmids or short hairpin RNA (shRNA). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to demonstrate the ERα bound to the half estrogen response element (half-ERE) located in PIWIL1 promoter. The expression of PIWIL1 and ERα in endometrial carcinoma tissues were investigated using immunohistochemistry and RT-qPCR. The proliferation ability of cancer cells were evaluated by MTT. Methylation status of the PIWIL1 promoter was detected by bisulfite sequencing PCR (BSP). Results In the present study, we found that PIWIL1 mediated E2-stimulated cancer cell proliferation. In ERα-positive endometrial cancer cells, we demonstrated that estrogen-ERα signaling significantly up-regulated the expression of PIWIL1, which was mediated by binding of the ERα onto the PIWIL1 promoter. Furthermore, we found that a half-ERE in the PIWIL1 promoter was essential for ERα binding. The PIWIL1 promoter was hypomethylated in ERα-positive endometrial cancer cells. Treatment with 5-aza-deoxycytidine (5-aza-dC) could up-regulate PIWIL1 expression. Conclusions These findings uncover a novel molecular mechanism by which estrogen-ERα signaling and DNA hypomethylation co-regulate PIWIL1 expression. These findings provide novel insights into the hormonal regulation of PIWIL1 in endometrial cancer and the PIWIL1’s role in estrogen-stimulated endometrial carcinogenesis.

OBJECTIVES: Intermittent calorie restriction has emerged as an effective alternative to continuous calorie restriction to impart favorable health benefits. However, there remains a degree of uncertainty about the optimal fasting length to simultaneously improve metabolic flexibility and promote positive behavior-change. While a 16-hr fast is commonly employed, it is possible that a shorter fast could result in similar metabolic benefits and better longer term adherence. Thus, we compared changes in hormone response between two 4-hr time intervals of fasting – 11–15 hrs. vs. 15–19 hrs. METHODS: Secondary analyses were performed from a 38-hr fasting study that enrolled 24 healthy, European men (18–30 y) with normal body mass index. Participants initiated a fast at 2300, arrived to the laboratory at 0800, and continued fasting in individual rooms. Venous blood was sampled at 13 time-points to measure insulin, glucose, adrenocorticotropin (ACTH), C-peptide, cortisol, and adiponectin via immunoassays. Data were normally distributed without outliers or missing values. Within-group differences were analyzed with paired t-tests. RESULTS: Insulin (pmol/L) declined between 11–15 hrs. (7.00; CI = 0.97, 13.02; P = .025), but not 15–19 hrs. (5.67; CI = –0.65, 11.98; P = .076). The decline in C-peptide (nmol/L) was sharper between 11–15 hrs. (0.09; CI = 0.05, 0.13; P = < .001), compared to 15–19 hrs. (0.06; CI = 0.02, 0.09; P = .003). Cortisol (nmol/L) also steeply declined between 11–15 hrs. (60.09; CI = 30.58, 89.59; P = < .001), but slightly increased between 15–19 hrs. (10.75; CI = −33.15, 11.65; P = 0.331). The change in glucose (mmol/L) was not different between 11–15 hrs. (0.02 mmol/L; CI = −0.15, 0.19; P = 0.784), but sharply decreased between 15–19 hrs. (0.24; CI = 0.10, 0.39; P = 0.002). No differences were detected for ACTH or adiponectin. CONCLUSIONS: Though substrate oxidation was not measured, changes in insulin, C-peptide, cortisol, and glucose were consistent with increased gluconeogenesis during 11–15 hrs. and lipid oxidation during 15–19 hrs. of fasting. Whereas cortisol often declines throughout the day, a slight increase was noted between 15–19 hrs. – evidence of a heightened sympathetic activity. Further study of hormone response during intermittent fasting will inform optimal length to promote health benefits and longer term adherence. FUNDING SOURCES: None

The prevalence of polycystic ovary syndrome (PCOS) has been gradually increasing among adult females worldwide. Laparoscopy drilling on ovary is the only available temporary solution with a high incidence of reoccurrence. S100A8 with S100A9 complex is believed to facilitate the cyst migration in PCOS condition. The high evident protein interaction network studies between PCOS biomarkers, cancer invasion markers, and the interactors of S100A8 confirm that this protein has strong interaction with other selective PCOS biomarkers, which may be associative in the immature cyst invasion process. Through the network studies, intensive structural and pathway analysis, S100A8 is identified as a targetable protein. In this research, the non-SELEX in silico method is adapted to construct RNA Library based on the consensus DNA sequence of Glucocorticoid Response Element (GRE) and screened the best nucleotide fragments which are bound within the active sites of the target protein. Selected sequences are joined as a single strand and screened the one which competitively binds with minimal energy. In vitro follow-up of this computational research, the designed RNA aptamer was used to infect the MCF7 cell line through Lipofectamine 2000 mediated delivery to study the anti-cell migration effect. Wound Scratch assay confirms that the synthesized 18-mer oligo has significant inhibition activity toward tumor cell migration at the cellular level.

Breast cancer is the most commonly diagnosed malignancy in female worldwide, over 70% of which are estrogen receptor α (ERα) positive. ERα has a crucial role in the initiation and progression of breast cancer and is an indicator of endocrine therapy, while endocrine resistance is an urgent problem in ER-positive breast cancer patients. In the present study, we identify a novel E3 ubiquitin ligase TRIM11 function to facilitate ERα signaling. TRIM11 is overexpressed in human breast cancer, and associates with poor prognosis. The protein level of TRIM11 is highly correlated with ERα. RNA-seq results suggest that ERα signaling may be an underlying target of TRIM11. Depletion of TRIM11 in breast cancer cells significantly decreases cell proliferation and migration. And the suppression effects can be reversed by overexpressing ERα. In addition, ERα protein level, ERα target genes expression and estrogen response element activity are also dramatically decreased by TRIM11 depletion. Further mechanistic analysis indicates that the RING domain of TRIM11 interacted with the N terminal of ERα in the cytoplasm and promotes its mono-ubiquitination, thus enhances ERα protein stability. Our study describes TRIM11 as a modulating factor of ERα and increases ERα stability via mono-ubiquitination. TRIM11 could be a promising therapeutic target for breast cancer treatment.

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers, which are approximately 5,000 basepairs apart, exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of the observed enhancer cooperativity. We generated genetic deletions of individual estrogen receptor α (ER) bound enhancers and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that a cluster of two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences between enhancers, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing active chromatin environment within an enhancer cluster is also needed in order for estrogen-driven gene regulation to occur.

Vascular inflammation is present in many cardiovascular diseases, and exogenous glucocorticoids have traditionally been used as a therapy to suppress inflammation. However, recent data have shown that endogenous glucocorticoids, acting through the endothelial glucocorticoid receptor, act as negative regulators of inflammation. Here, we performed ChIP for the glucocorticoid receptor, followed by next-generation sequencing in mouse endothelial cells to investigate how the endothelial glucocorticoid receptor regulates vascular inflammation. We identified a role of the Wnt signaling pathway in this setting and show that loss of the endothelial glucocorticoid receptor results in upregulation of Wnt signaling both in vitro and in vivo using our validated mouse model. Furthermore, we demonstrate glucocorticoid receptor regulation of a key gene in the Wnt pathway, Frzb, via a glucocorticoid response element gleaned from our genomic data. These results suggest a role for endothelial Wnt signaling modulation in states of vascular inflammation.

Objective: Primary generalized glucocorticoid resistance (PGGR) is a rare hereditary disease characterized by generalized partial target-tissue insensitivity to glucocorticoids. To date, few cases have been reported, and more cases, especially involving other races, are needed to fully understand this disease. Methods: This study presented a novel glucocorticoid receptor mutation in a PGGR pedigree. The index patient was a 14-year-old male with fatigue, hypokalemia, hypertension, and polyuria. Eleven family members were available for the genetic screen. Next-generation sequencing and Sanger sequencing were used to identify the mutation. We systematically investigated the molecular mechanism through which the mutation impaired glucocorticoid signal transduction in COS-7 cells. Results: The index patient carried a de novo homo-zygous mutation within exon 6 (c.1652C>A, p.551S>Y), whereas eight family members carrying a heterozygous mutation were all phenotypically silent. The affinity of the human glucocorticoid receptor (hGR) for the ligand was 1.97-fold lower in the patient than in the family members. Mutant hGRα (551Y) displayed a 3.2-fold reduction in its ability to transactivate glucocorticoid-responsive genes. When exposed to the same concentration of dexamethasone, hGRα (551Y) displayed a reduced ability to trans-locate into the nucleus and decreased levels of hGR dimer formation and could not effectively induce the glucocorticoid response element to regulate the transcription of related genes. After 2 years of dexamethasone treatment, the volume of the left and right adrenal glands of the index subject decreased by 55.6% and 32.4%, respectively. The pituitary volume decreased by 18.9%. During the 2-year follow-up, none of the heterozygous carriers developed hypertension or hypokalemia. Conclusion: We described a novel homozygous glucocorticoid receptor mutation causing PGGR. This homozygous mutation leads to hypertension and hypokalemia, but its heterozygous mutation has no relevant clinical symptoms. Abbreviations: ACTH = adrenocorticotropic hormone; DBD = DNA-binding domain; GR = glucocorticoid receptor; GRE = glucocorticoid response element; hGR = human glucocorticoid receptor; LBD = ligand-binding domain; PGGR = primary generalized glucocorticoid resistance.

This paper reports that the human peroxisomal 3-ketoacyl-CoA thiolase expression shows three transcripts: Tr1 (1705 bp), Tr2 (1375 bp) and Tr3 (1782 bp). Their highest expression is observed in the human liver and at a lesser extent in hepatic-derived HepG2 cells. The intestine and blood and endothelial cells show lower expression. The lowest expression is found in adipocytes. The transcript Tr3 appears to be the most abundant. So far, no data have been published regarding the regulation of the human peroxisomal thiolase. After cloning a fragment of the 5′ region involved in the regulation of the human thiolase gene, the effects of different treatments have been studied on the thiolase expression in the hepatoma HepG2 human cell line. Biocomputing analysis indicates that (i) a GRE (glucocorticoid response element) is located at −650 bp upstream of the transcription initiation site; (ii) a C/EBPα (CCAAT/enhancer-binding protein) binding site is located at − 1000 bp upstream of the transcription initiation site – and (iii) there is no putative PPRE (peroxisome proliferator-activated receptor response element). In the human HepG2 cells, thiolase expression is upregulated by glucose and downregulated by insulin and sterols, while dexamethasone and fatty acids have no effect. The ciprofibrate, a peroxisome proliferator, leads only to a weak stimulation of the mRNA expression as compared to thiolase B expression in the rat liver.

17β-estradiol (E2) is considered to be an important instigator of prolactinoma, and can positively regulate the expression of calbindin-D9k (CaBP-9k) which contains an estrogen responsive element (ERE) via estrogen receptors (ERs). However, the detailed mechanism of E2 in promoting CaBP-9k expression and their roles in prolactinoma progression remain unclear. Here, we aimed to characterize it. The luciferase gene reporter assay with luc-ERE transfection showed that E2 treatment significantly enhanced the transcriptional level of CaBP-9k, whereas CaBP-9k activity was reduced when GH3 and MMQ cells were treated with AZD9496, an antagonist of ERα. E2 treatment increased the protein expressions of CaBP-9k and ERα but not ERβ, whereas this effect was also abolished when cells were treated with AZD9496. Besides, immunoprecipitation (IP) and immunofluorescence assays demonstrated that CaBP-9k could directly interact with ERα not ERβ, and Chromatin IP (ChIP) assay showed that ERα could bind to ERE of the CaBP-9k promoter. Moreover, cell counting kit-8 (CCK-8) and flow cytometry assays showed that E2 treatment significantly enhanced cell viability and inhibited cell apoptosis, but these effects were all abolished when ERα was down-regulated by short hairpin RNA (shRNA) or inhibited by AZD9496, as well as CaBP-9K suppression in both GH3 and MMQ cell lines. Taken together, these findings indicated that E2 stimulation promoted prolactin cell proliferation and inhibited cell apoptosis through ERα-induced CaBP-9k up-regulation, which then accelerated the advanced progression of prolactinoma.

P21-activated kinase 1 (Pak1) is known to be involved in a plethora of functions including cell growth, survival and can lead to cell transformation and tumor progression especially in breast tissue. Multiple studies have shown Pak1 dysregulation as a change in DNA copy number as well as gene expression levels, suggesting many regulatory mechanisms at transcriptional and translational level. However, very little is known about the transcriptional regulation of the human Pak1 promoter. Here, we focus on Pak1 promoter regulation by steroid hormones along with their respective receptors that are also crucial players in breast tissue function and tumorigenesis. Our results show high Pak1 expression in breast cancer cell lines and in breast tumor tissue. It also suggests that Pak1 is hormone responsive, whose expression can be modulated by steroid hormones namely, estrogen in the form of 17β-estradiol (E2) and progesterone (P4). Sequence analysis of a 3.2kb Pak1 proximal promoter region shows the presence of PRE (progesterone response element) and ERE (estrogen response element) half sites, that were further cloned and characterized. Results from promoter analysis showed that Pak1 promoter activity is mediated by PR via its binding to PRE present on the Pak1 promoter that was further reaffirmed in vitro by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results together suggest that it is the PR isoform B regulates Pak1 promoter. To our knowledge, this is the first study to report the detailed characterization and transcriptional regulation of the human Pak1 promoter by steroid hormones.

In ovarian follicle development, estrogen acts as a regulatory molecule to mediate proliferation and differentiation of follicular cells. ERα (estrogen receptor α) exerts regulatory function classically by binding directly to the estrogen response element, recruiting co-factors and activating or repressing transcription in response to E2. In this study, we used ChIP-seq to map ERα-binding sites in ovaries of Hy-line Brown commercial hens at 45d, 90d and 160d. In total, 24,886, 21,680 and 23,348 binding sites were identified in the ovaries of hens at 45d, 90d and 160d, which are linked to 86, 83 and 74 genes, respectively. The PPI network contains 47 protein nodes and 164 interaction edges, among which, AKT1 (V-Akt Murine Thymoma Viral Oncogene Homolog 1) and ACTN2 (Actinin Alpha 2) with the highest weight in the network, followed by CREB1 (CAMP Responsive Element Binding Protein 1), and EPHA5 (EPH Receptor A5) were identified. These genes are likely related to sexual maturity in hens. This study also provides insight into the regulation of the ERα target gene networks and a reference for understanding ERα-regulated transcription.