In mouse ovaries, liver receptor homolog-1 [nuclear receptor subfamily 5, group A, member 2 (Nr5a2)] expression is restricted to granulosa cells. Mice with Nr5a2 depletion in this cell population fail to ovulate. To determine whether Nr5a2 is essential for granulosa cell proliferation during follicular maturation, we generated granulosa-specific conditional knockout mice (genotype Nr5a2 floxed Cre-recombinase driven by the anti-Müllerian type II receptor, hereafter cKO) with Nr5a2 depletion from primary follicles forward. Proliferation in cKO granulosa cells was substantially reduced relative to control (CON) counterparts, as assessed by bromodeoxyuridine incorporation, proliferative cell nuclear antigen expression, and fluorescent-activated cell sorting. Microarray analysis revealed >2000 differentially regulated transcripts between cKO and CON granulosa cells. Major gene ontology pathways disrupted were proliferation, steroid biosynthesis, female gamete formation, and ovulatory cycle. Transcripts for key cell-cycle genes, including Ccnd1, Ccnd2, Ccne1, Ccne2, E2f1, and E2f2, were in reduced abundance. Transcripts from other cell-cycle-related factors, including Cdh2, Plagl1, Cdkn1a, Prkar2b, Gstm1, Cdk7, and Pts, were overexpressed. Although the follicle-stimulating hormone and estrogen receptors were overexpressed in the cKO animals, in vivo treatment with estradiol-17β failed to rescue decreased proliferation. In vitro inactivation of Nr5a2 using the ML180 reverse agonist similarly decreased cell-cycle-related gene transcripts and downstream targets, as in cKO mice. Pharmacological inhibition of β-catenin, an Nr5a2 cofactor, decreased cyclin gene transcripts and downstream targets. Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling immunofluorescence and quantitative polymerase chain reaction of pro/antiapoptotic and autophagic markers showed no differences between cKO and CON granulosa cells. Thus, Nr5a2 is essential for granulosa cell proliferation, but its depletion does not alter the frequency of apoptosis nor autophagy.

Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily. Here, we show that genetic ablation of Nr2f6 significantly improves survival in the murine transgenic TRAMP prostate cancer model. Furthermore, Nr2f6(-/-) mice spontaneously reject implanted tumors and develop host-protective immunological memory against tumor rechallenge. This is paralleled by increased frequencies of both CD4(+) and CD8(+) T cells and higher expression levels of interleukin 2 and interferon γ at the tumor site. Mechanistically, CD4(+) and CD8(+) T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds. Adoptive transfer of Nr2f6-deficient T cells into tumor-bearing immunocompetent mice is sufficient to delay tumor outgrowth. Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) is a member of the steroid/thyroid hormone receptor superfamily with a crucial role in organogenesis, angiogenesis, cardiovascular development and tumorigenesis. However, there is limited knowledge about the cistrome and transcriptome of NR2F2 in breast cancer. In this study, we mapped the regulatory mechanism by NR2F2 using functional genomic methods. To investigate the clinical significance of NR2F2 in breast cancer, The Cancer Genome Atlas (TCGA) data were used. These results show that a high NR2F2 is associated with better survival of a specific subset of patients, namely those with luminal A breast cancer. Therefore, genome-wide NR2F2 and estrogen receptor alpha (ERα) binding sites were mapped in luminal A breast cancer cells using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), revealing that most NR2F2 overlap with ERα that are co-occupied by forkhead box A1 (FOXA1) and GATA binding protein 3 (GATA3) in active enhancer regions. NR2F2 overlaps with highly frequent ERα chromatin interactions, which are essential for the formation of ERα-bound super-enhancers. In the process of the transcriptome profiling of NR2F2-depleted breast cancer cells such differentially expressed genes have been identified that are involved in endocrine therapy resistance and are also ERα target genes. Overall, these findings demonstrate that the NR2F2 nuclear receptor has a key role in ERα-mediated transcription and it can offer a potential therapeutic target in patients with luminal A breast cancer.

Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.

Glucocorticoids act through the glucocorticoid receptor (GR) encoded by the nuclear receptor subfamily 3 group C member 1 (NR3C1) gene.

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Nuclear receptor subfamily 1, group I, member 2 (NR1I2), commonly known as steroid and xenobiotic receptor (SXR) in humans, is a key ligand-dependent transcription factor responsible for the regulation of xenobiotic, steroid, and bile acid metabolism. The ligand-binding domain is principally responsible for species-specific activation of NR1I2 in response to xenobiotic exposure.

Oxidative stress activates macroautophagy/autophagy and contributes to atherogenesis via lipophagic flux, a form of lipid removal by autophagy. However, it is not known exactly how endogenous antioxidant enzymes are involved in lipophagic flux. Here, we demonstrate that the antioxidant PRDX1 (peroxiredoxin 1) has a crucial role in the maintenance of lipophagic flux in macrophages. PRDX1 is more highly expressed than other antioxidant enzymes in monocytes and macrophages. We determined that Prdx1 deficiency induced excessive oxidative stress and impaired maintenance of autophagic flux in macrophages. Prdx1-deficient macrophages had higher intracellular cholesterol mass and lower cholesterol efflux compared with wild type. This perturbation in cholesterol homeostasis was due to impaired lipophagic cholesterol hydrolysis caused by excessive oxidative stress, resulting in the inhibition of free cholesterol formation and the reduction of NR1H3 (nuclear receptor subfamily 1, group H, member 3) activity. Notably, impairment of both lipophagic flux and cholesterol efflux was restored by the 2-Cys PRDX-mimics ebselen and gliotoxin. Consistent with this observation, apoe -/- mice transplanted with bone marrow from prdx1-/-apoe-/- mice had increased plaque formation compared with apoe-/- BM-transplanted recipients. This study reveals that PRDX1 is crucial to regulating lipophagic flux and maintaining macrophage cholesterol homeostasis against oxidative stress. We suggest that PRDX1-dependent control of oxidative stress may provide a strategy for treating atherosclerosis and autophagy-related human diseases.

Early life stress has been linked to increased methylation of the Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) gene, which codes for the glucocorticoid receptor. Moreover, early life stress has been associated with substance use initiation at a younger age, a risk factor for developing substance use disorders. However, no studies to date have investigated whether NR3C1 methylation can predict substance use in young individuals. This study included adolescents 13-14 years of age that reported no history of substance use at baseline, (N = 1041; males = 46%). Participants contributed saliva DNA samples and were followed in middle adolescence as part of KUPOL, a prospective cohort study of 7th-grade students in Sweden. Outcome variables were self-reports of (i) recent use, (ii) lifetime use, and (iii) use duration of (a) alcohol, (b) tobacco products, (c) cannabis, or (d) any substance. Outcomes were measured annually for three consecutive years. The predictor variable was DNA methylation at the exon 1 F locus of NR3C1. Risk and rate ratios were calculated as measures of association, with or without adjustment for internalizing symptoms and parental psychiatric disorders. For a subset of individuals (N = 320), there were also morning and afternoon salivary cortisol measurements available that were analyzed in relation to NR3C1 methylation levels. Baseline NR3C1 hypermethylation associated with future self-reports of recent use and use duration of any substance, before and after adjustment for potential confounders. The overall estimates were attenuated when considering lifetime use. Sex-stratified analyses revealed the strongest association for cigarette use in males. Cortisol analyses revealed associations between NR3C1 methylation and morning cortisol levels. Findings from this study suggest that saliva NR3C1 hypermethylation can predict substance use in middle adolescence. Additional longitudinal studies are warranted to confirm these findings.

The downregulation of farnesoid X receptor (FXR; gene name, nuclear receptor subfamily 1 group h member 4), an enteric nuclear bile acid receptor, has been reported in colorectal carcinoma (CRC), and FXR expression has been inversely correlated with CRC stage and clinical outcome. FXR knockdown in chronic colitis mouse models of intestinal tumorigenesis results in early mortality and increased tumor progression via promoting Wnt signaling. The aim of the present study was to explore the effects and mechanism of FXR on the Wnt/β-catenin signal pathway in CRC. FXR and β-catenin protein expression levels were detected in an ulcerative colitis mouse model and human colon cancer cell lines (HT-29, Caco-2 and HCT-116). Gain- and loss-of-function studies were conducted by transfecting colon cancer cells with FXR siRNA and treating them with the FXR agonist GW4064. Subsequently, β-catenin transcriptional activity was measured using the dual-luciferase assay, and β-catenin/TCF4 complex levels and β-catenin protein and mRNA expression levels were determined. FXR and β-catenin expression levels were inversely associated in both the animal model and colon cancer cells. The Wnt signaling pathway was activated by increased β-catenin/TCF4 complex levels upon FXR silencing; however, mRNA and protein levels of β-catenin were not significantly affected. The FXR agonist GW4064 significantly inhibited the proliferation of cells but promoted the transcriptional activity of β-catenin. Thus, the present study demonstrated that FXR influences the Wnt/β-catenin signaling pathway. Furthermore, loss of FXR expression promotes the transcriptional activity of β-catenin, whereas FXR activation results in the opposite effect.

Radiation therapy (RT) is widely applied in cancer treatment. The sensitivity of tumor cells to RT is the key to the treatment. This study probes the role and mechanism of miR-20b-5p in Pembrolizumab's affecting the radiosensitivity of tumor cells. After Pembrolizumab treatment or cell transfection (miR-20b-5p mimics and miR-20b-5p inhibitors), tumor cells (NCI-H460 and ZR-75-30) were exposed to RT. The sensitivity of NCI-H460 and ZR-75-30 to RT was evaluated by monitoring cell proliferation and apoptosis. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were adopted to evaluate the binding relationship between miR-20b-5p and CD274 (PD-L1). The xenograft model was established in nude mice to examine the mechanism of action of Pembrolizumab in vivo. Our outcomes exhibited that either Pembrolizumab treatment or miR-20b-5p overexpression potentiated radiosensitivity of tumor cells. Overexpressing miR-20b-5p enhanced radiosensitization of Pembrolizumab in vivo and in vitro by targeting PD-L1 and inactivating PD-L1/PD1. Overall, miR-20b-5p overexpression combined with Pembrolizumab potentiated cancer cells' sensitivity to RT by repressing PD-L1/PD1.Abbreviations Akt: serine/threonine kinase 1; cDNA: complementary DNA; CO2: carbon dioxide; EDTA: Ethylene Diamine Tetraacetic Acid; ENCORI: The Encyclopedia of RNA Interactomes; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IGF2BP2: insulin like growth factor 2 mRNA binding protein 2; IHC: Immunohistochemistry; LncRNA MALAT1: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1; miRNAs: MicroRNAs; Mt: Mutant type; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; NC: negative control; NR2F2: nuclear receptor subfamily 2 group F member 2; NSCLC: non-small cell lung cancer; OD: optical density; PBS: phosphate-buffered saline; PD-L1: Programmed death-ligand 1; PD-1: programmed death 1; PI3K: phosphatidylinositol 3-kinase; qRT-PCR: Quantitative reverse transcription-polymerase chain reaction; RIP: RNA immunoprecipitation; RIPA: Radio Immunoprecipitation Assay; RRM2: ribonucleotide reductase regulatory subunit M2; RT: Radiation therapy; U6: U6 small nuclear RNA; V: volume; WB: Western blot; Wt: wild type; x ± sd: mean ± standard deviation.

The functions of genes involved in idiopathic portal hypertension (IPH) remain unidentified. The present study was undertaken to identify the functions of genes expressed in blood samples from patients with IPH through comprehensive analysis of gene expression using DNA microarrays. The data were compared with data from healthy individuals to explore the functions of genes showing increased or decreased expression in patients with IPH. In cluster analysis, no dominant probe group was shown to differ between patients with IPH and healthy controls. In functional annotation analysis using the Database for Annotation Visualization and Integrated Discovery tool, clusters showing dysfunction in patients with IPH involved gene terms related to the immune system. Analysis using network-based pathways revealed decreased expression of adenosine deaminase, ectonucleoside triphosphate diphosphohydrolase 4, ATP-binding cassette, subfamily C, member 1, transforming growth factor-β, and prostaglandin E receptor 2; increased expression of cytochrome P450, family 4, subfamily F, polypeptide 3, and glutathione peroxidase 3; and abnormalities in the immune system, nucleic acid metabolism, arachidonic acid/leukotriene pathways, and biological processes. These results suggested that IPH involved compromised function of immunocompetent cells and that such dysfunction may be associated with abnormalities in nucleic acid metabolism and arachidonic acid/leukotriene-related synthesis/metabolism.

The role of inflammation in mood disorders has received increased attention. There is substantial evidence that cytokine therapies, such as interferon alpha (IFN-alpha), can induce depressive symptoms. Indeed, proinflammatory cytokines change brain function in several ways, such as altering neurotransmitters, the glucocorticoid axis, and apoptotic mechanisms. This study aimed to evaluate the impact on mood of initiating IFN-alpha and ribavirin treatment in a cohort of patients with chronic hepatitis C. We investigated clinical, personality, and functional genetic variants associated with cytokine-induced depression.

There are several indications that malfunctions of the circadian clock contribute to depression. To search for particular circadian gene polymorphisms associated with depression, diverse polymorphisms were genotyped in two samples covering a range of depressed volunteers and participants with normal mood.

Hypoxia is an important feature for the progression of hepatocellular carcinoma (HCC). Long noncoding RNA nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) is dysregulated in HCC. However, the role and mechanism of N2RF1-AS1 in hypoxia-induced glycolysis and migration remain unclear.

Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation.

Early weaning induces intestinal injury, leading to a series of long-term symptoms such as inflammation, malabsorption and diarrhea. In this study, we hypothesized that microbes and their metabolites modulate the host's inflammatory response to early weaning stress in a goat model. A total of 18 female Tibetan goat kids (n = 9) were weaned from their mothers at 28 d (D28) and 60 d (D60) postpartum. D60 and D28 groups were fed the same solid diet ad libitum from weaning to 75 d of age. The colonic epithelium was subject to RNA-sequencing, the caecal digesta metabolomics were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the caecal microbiota composition was analysed by 16S ribosomal RNA gene sequencing. We found that early weaning substantially increased the colonic pro-apoptotic gene expression of B-cell lymphoma associated X (Bax), caspase-9, and caspase-3, and decreased the expression of zonula occludens-1 (ZO-1) and claudin-1 (P < 0.01). In addition, a significant Bacteroides acidifaciens enrichment was observed in the hindgut of early-weaned goats (P < 0.01), which negatively correlated with lysophosphatidylcholine products. Similarly, the chemokine signaling, IL-17 signaling, and peroxisome proliferators-activated receptor (PPAR) signaling pathways were upregulated in the colonic mucosa of the early-weaned goats. By applying caecal microbiota transplantation from goats to defaunated C57/6J mice, we confirmed that caecal microbiota of D28 goat kids increased the relative abundance of B. acidifaciens and significantly up-regulated the genes of Bax, G protein-coupled receptor (GPR) 109A, GPR 43, fatty acid binding protein 6, nuclear receptor subfamily 1 group H member 3, angiotensin converting enzyme 2, and IL-6 expression (P < 0.05), and decreased ZO-1, and claudin-1 protein expression in the mice jejunum and colon (P < 0.001). These results proposed that the hindgut microbiota and metabolites mediate the barrier function weakening during early weaning, and the relative abundance of B. acidifaciens was negatively correlated with the hindgut barrier gene expression. This study demonstrates how weaning stress can affect key host-microbe interaction regulators in the hindgut, in a lysophosphatidylcholine dependent and independent manner. Furthermore, based on our mice data, these results are transferable to other mammal species.

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3β-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

While it has been hypothesized that brown adipocytes responsible for mammalian thermogenesis are absent in birds, the existence of beige fat has yet to be studied directly. The present study tests the hypothesis that beige fat emerges in birds as a mechanism of physiological adaptation to cold environments. Subcutaneous neck adipose tissue from cold-acclimated or triiodothyronine (T3)-treated chickens exhibited increases in the expression of avian uncoupling protein (avUCP, an ortholog of mammalian UCP2 and UCP3) gene and some known mammalian beige adipocyte-specific markers. Morphological characteristics of white adipose tissues of treated chickens showed increased numbers of both small and larger clusters of multilocular fat cells within the tissues. Increases in protein levels of avUCP and mitochondrial marker protein, voltage-dependent anion channel, and immunohistochemical analysis for subcutaneous neck fat revealed the presence of potentially thermogenic mitochondria-rich cells. This is the first evidence that the capacity for thermogenesis may be acquired by differentiating adipose tissue into beige-like fat for maintaining temperature homeostasis in the subcutaneous fat 'neck warmer' in chickens exposed to a cold environment.

The failure of treatment for breast cancer usually results from distant metastasis in which the epithelial-mesenchymal transition (EMT) plays a critical role. Hyperinsulinemia, the hallmark of Type 2 diabetes mellitus (T2DM), has been regarded as a key risk factor for the progression of breast cancer. Nuclear receptor subfamily 2, group F, member 2 (NR2F2) has been implicated in the development of breast cancer, however its contribution to insulin-induced EMT in breast cancer remains unclear.