Carbamate Pesticides: Shedding Light on Their Impact on the Male Reproductive System.

Carbamates are widely used and known around the world as pesticides in spite of also having medical applications. This class of chemicals is classified as acetylcholinesterase inhibitors, blocking acetylcholine hydrolyzation in a reversible manner. Their lack of species selectivity and their reported high toxicity can induce, upon exposure, adverse outcomes in male fertility that may lead to infertility. In addition, they are also considered endocrine-disrupting chemicals and can interfere with the hypothalamic-pituitary-testicular axis, essential for the normal function of the male reproductive system, thus being able to provoke male reproductive dysfunctions. Although the molecular mechanisms are not fully understood, various signaling pathways, such as those mediated by acetylcholine or kisspeptin, are affected by exposure to carbamates, thus compromising steroidogenesis and spermatogenesis. Over the last decades, several studies, both in vitro and in vivo, have reported a myriad of negative effects of carbamates on the male reproductive system. In this review, an up-to-date overview of the impact of carbamates on the male reproductive system is discussed, with an emphasis on the role of these compounds on acetylcholine regulation and the male endocrine system.

The orphan nuclear receptor NR0B2 could be a novel susceptibility locus associated with microsatellite-stable, APC mutation-negative early-onset colorectal carcinomas with metabolic manifestation.

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-ß, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

[Suppression of NR0B2 gene in Clear Cell Renal Cell Carcinoma Is Associated with Hypermethylation of Its Promoter].

Clear cell renal cell carcinoma (ccRCC) is a common urologic malignancy. Understanding of the transcriptional regulation of oncogenes and tumor suppressor genes involved is critical for the development of the treatments for renal tumors. Using ccRCC subdivision of the TCGA dataset, we identified NR0B2 encoding orphan nuclear receptor as a tumor suppressor candidate in renal tissue. In independent cohort of primary renal tumors, quantitative PCR experiments confirmed significant suppression of NR0B2 mRNA in 86% of ccRCC samples studied. In 80% of these cases, we detected the hypermethylation of the NR0B2 pro-moter region. These results suggest that NR0B2 is a tumor suppressor gene in ccRCC, and that the hypermethylation of promoter region is the main mechanism of its downregulation.

Circadian rhythmicity: A functional connection between differentiated embryonic chondrocyte-1 (DEC1) and small heterodimer partner (SHP).

Circadian rhythm misalignment has been increasingly recognized to pose health risk for a wide range of diseases, particularly metabolic disorders. The liver maintains metabolic homeostasis and expresses many circadian genes, such as differentiated embryo chondrocyte-1 (DEC1) and small heterodimer partner (SHP). DEC1 is established to repress transcription through E-box elements, and SHP belongs to the superfamily of nuclear receptors and has multiple E-box elements in its promoter. Importantly, DEC1 and SHP are inversely oscillated. This study was performed to test the hypothesis that the SHP gene is a target gene of DEC1. Cotransfection demonstrated that DEC1 repressed the SHP promoter and attenuated the transactivation of the classic circadian activator complex of Clock/Bmal1. Site-directed mutagenesis, electrophoretic mobility shift assay and chromatin immunoprecipitation established that the repression was achieved through the E-box in the proximal promoter. Transfection of DEC1 suppressed the expression of SHP. In circadian-inducing cells, the epileptic agent valproate inversely altered the expression of DEC1 and SHP. Both DEC1 and SHP are involved in energy balance and valproate is known to induce hepatic steatosis. Our findings collectively establish that DEC1 participates in the negative loop of SHP oscillating expression with potential implications in metabolic homeostasis.

Identification and Characteristics of Novel Mutations in Nonsyndromic Monogenic Obesity.

Nonsyndromic monogenic obesity (NSMO) is a class of individual obesity that is independent of the environment and caused by a single gene mutation. It is mostly caused by mutations in LEP, LEPR, PCSK1, as well as some rare mutations in UCP3, NR0B2, and PPARG. Among 30 obesity patients, five patients are identified with positive gene detection. For the first time, the c.624C>T mutation associated with PCSK1, and the c.50G>A and c.293_301delinsAC mutations associated with NR0B2, as well as the obesity phenotype mutation (c.284A>G) associated with PPARG is confirmed. Following this, the genotype-clinical phenotype, mutation hotspots, and mutation distributions of each gene are summarized, and the genetic characteristics of NSMO are analyzed. The locations of mutation c.50G>A, and c.284A>G are highly conserved according to the sequencing alignment. According to the findings, the c.624C>T mutation in PCSK1 is a newly discovered synonymous mutation, but it can result in significant early-onset obesity. Additionally, the mutation of c.284A>G(PPARG) can lead to a variety of clinical phenotypes and the mutation of UCP3 and NR0B2 may increase the risk of type 2 diabetes mellitus. This study enriches the human NSMO gene mutation database and provides a scientific basis for clinically accurate diagnosis and treatment.

Paradoxical feeding activation of gut lipophagy by FGF15/FGF19-NR0B2/SHP-TFEB.

Macroautophagic/autophagic degradation of lipid droplets, lipophagy, is activated by fasting but repressed by feeding. Surprisingly, our recent study showed that this is not the case in the gut, where feeding activates lipophagy, reducing intestinal lipid levels. Transgenic mouse studies revealed that feeding activation of gut lipophagy requires both FGF15/FGF19 (fibroblast growth factor 15/fibroblast growth factor 19) and an orphan nuclear receptor, NR0B2/SHP (nuclear receptor subfamily 0, group B, member 2). Mechanistically, feeding-induced FGF15/FGF19 activates intestinal PRKC/PKC signaling, which in turn phosphorylates NR0B2 and the autophagic activator TFEB (transcription factor EB), leading to their nuclear localization and transcriptional induction of lipophagy network genes, including Ulk1 and Pnpla2/Atgl. Given that an essential function of the gut is to distribute dietary lipids throughout the body, this study identifies a physiologically important homeostatic mechanism to maintain healthy lipid levels. The intestinal FGF15/FGF19-NR0B2/SHP-TFEB pathway that regulates postprandial lipids by lipophagic activation, thus, may provide novel targets for treating dyslipidemia and obesity.

The Orphan Nuclear Receptor Gene NR0B2 Is a Favorite Prognosis Factor Modulated by Multiple Cellular Signal Pathways in Human Liver Cancers.

BACKGROUND: Liver cancer is a leading cause of cancer death worldwide, and novel prognostic factor is needed for early detection and therapeutic responsiveness monitoring. The orphan nuclear receptor NR0B2 was reported to suppress liver cancer development in a mouse model, and its expression levels were reduced in liver cancer tissues and cell lines due to hypermethylation within its promoter region. However, it is not clear if NR0B2 expression is associated with cancer survival or disease progression and how NR0B2 gene expression is regulated at the molecular level. METHODS: Multiple cancer databases were utilized to explore NR0B2 gene expression profiles crossing a variety of human cancers, including liver cancers, on several publicly assessable bioinformatics platforms. NR0B2 gene expression with or without kinase inhibitor treatment was analyzed using the qPCR technique, and NR0B2 protein expression was assessed in western blot assays. Two human hepatocellular carcinoma cell lines HepG2 and Huh7, were used in these experiments. NR0B2 gene activation was evaluated using NR0B2 promoter-driven luciferase reporter assays. RESULTS: NR0B2 gene is predominantly expressed in liver tissue crossing human major organs or tissues, but it is significantly downregulated in liver cancers. NR0B2 expression is mostly downregulated in most common cancers but also upregulated in a few intestinal cancers. NR0B2 gene expression significantly correlated with patient overall survival status in multiple human malignancies, including lung, kidney, breast, urinary bladder, thyroid, colon, and head-neck cancers, as well as liposarcoma and B-cell lymphoma. In liver cancer patients, higher NR0B2 expression is associated with favorite relapse-free and progression-free survival, especially in Asian male patients with viral infection history. In addition, NR0B2 expression negatively correlated with immune infiltration and PIK3CA and PIK3CG gene expression in liver cancer tissues. In HepG2 and Huh7 cells, NR0B2 expression at the transcription level was drastically reduced after MAPK inhibition but was significantly enhanced after PI3K inhibition. CONCLUSION: NR0B2 gene expression is altered mainly in most human malignancies and significantly reduced in liver cancers. NR0B2 is a prognosis factor for patient survival in liver cancers. MAPK and PI3K oppositely modulate NR0B2 expression, and NR0B2 gene upregulation might serve as a therapeutic responsiveness factor in anti-PI3K therapy for liver cancer.

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors.

Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

Interleukin-10 Attenuates Hypochlorous Acid-Mediated Cytotoxicity to HEI-OC1 Cochlear Cells.

Inflammatory reaction plays a crucial role in the pathophysiology of acquired hearing loss such as ototoxicity and labyrinthitis. In our earlier work, we showed the pivotal role of otic fibrocytes in cochlear inflammation and the critical involvement of proinflammatory cytokines in cisplatin ototoxicity. We also demonstrated that otic fibrocytes inhibit monocyte chemoattractant protein 1 (CCL2) upregulation in response to interleukin-10 (IL-10) via heme oxygenase 1 (HMOX1) signaling, resulting in suppression of cochlear inflammation. However, it is still unclear how IL-10 affects inflammation-mediated cochlear injury. Here we aim to determine how hypochlorous acid, a model inflammation mediator affects cochlear cell viability and how IL-10 affects hypochlorous acid-mediated cochlear cell injury. NaOCl, a sodium salt of hypochlorous acid (HOCl) was found to induce cytotoxicity of HEI-OC1 cells in a dose-dependent manner. Combination of hydrogen peroxide and myeloperoxidase augmented cisplatin cytotoxicity, and this synergism was inhibited by N-Acetyl-L-cysteine and ML-171. The rat spiral ligament cell line (RSL) appeared to upregulate the antioxidant response element (ARE) activities upon exposure to IL-10. RSL cells upregulated the expression of NRF2 (an ARE ligand) and NR0B2 in response to CoPP (a HMOX1 inducer), but not to ZnPP (a HMOX1 inhibitor). Adenovirus-mediated overexpression of NR0B2 was found to suppress CCL2 upregulation. IL-10-positive cells appeared in the mouse stria vascularis 1 day after intraperitoneal injection of lipopolysaccharide (LPS). Five days after injection, IL-10-positive cells were observed in the spiral ligament, spiral limbus, spiral ganglia, and suprastrial area, but not in the stria vascularis. IL-10R1 appeared to be expressed in the mouse organ of Corti as well as HEI-OC1 cells. HEI-OC1 cells upregulated Bcl-xL expression in response to IL-10, and IL-10 was shown to attenuate NaOCl-induced cytotoxicity. In addition, HEI-OC1 cells upregulated IL-22RA upon exposure to cisplatin, and NaOCl cytotoxicity was inhibited by IL-22. Taken together, our findings suggest that hypochlorous acid is involved in cochlear injury and that IL-10 potentially reduces cochlear injury through not only inhibition of inflammation but also enhancement of cochlear cell viability. Further studies are needed to determine immunological characteristics of intracochlear IL-10-positive cells and elucidate molecular mechanisms involved in the otoprotective activity of IL-10.

Diet-dependent retinoid effects on liver gene expression include stellate and inflammation markers and parallel effects of the nuclear repressor Shp.

For mice, a maternal vitamin A (VA)-deficient diet initiated from midgestation (GVAD) produces serum retinol deficiency in mature offspring. We hypothesize that the effects of GVAD arise from preweaning developmental changes. We compare the effect of this GVAD protocol in combination with a postweaning high-fat diet (HFD) or high-carbohydrate diet (LF12). Each is compared to an equivalent VA-sufficient combination. GVAD extensively decreased serum retinol and liver retinol, retinyl esters, and retinoid homeostasis genes (Lrat, Cyp26b1 and Cyp26a1). These suppressions were each more effective with LF12 than with HFD. Postweaning initiation of VA deficiency with LF12 depleted liver retinoids, but serum retinol was unaffected. Liver retinoid depletion, therefore, precedes serum attenuation. Maternal LF12 decreased the obesity response to the HFD, which was further decreased by GVAD. LF12 fed to the mother and offspring extensively stimulated genes marking stellate activation (Col1a1, Timp2 and Cyp1b1) and novel inflammation markers (Ly6d, Trem2 and Nupr1). The GVAD with LF12 diet combination suppressed these responses. GVAD in combination with the HFD increased these same clusters. A further set of expression differences on the HFD when compared to a high-carbohydrate diet was prevented when GVAD was combined with HFD. Most of these GVAD gene changes match published effects from deletion of Nr0b2/Shp, a retinoid-responsive, nuclear co-repressor that modulates metabolic homeostasis. The stellate and inflammatory increases seen with the high-carbohydrate LF12 diet may represent postprandial responses. They depend on retinol and Shp, but the regulation reverses with an HFD.

Modulation of expression of the nuclear receptor NR0B2 (small heterodimer partner 1) and its impact on proliferation of renal carcinoma cells.

Mammalian nuclear receptors (NRs) are transcription factors regulating the expression of target genes that play an important role in drug metabolism, transport, and cellular signaling pathways. The orphan and structurally unique receptor small heterodimer partner 1 (syn NR0B2) is not only known for its modulation of drug response, but has also been reported to be involved in hepatocellular carcinogenesis. Indeed, previous studies show that NR0B2 is downregulated in human hepatocellular carcinoma, suggesting that NR0B2 acts as a tumor suppressor via inhibition of cellular growth and activation of apoptosis in this tumor entity. The aim of our study was to elucidate whether NR0B2 may also play a role in other tumor entities. Comparing NR0B2 expression in renal cell carcinoma and adjacent nonmalignant transformed tissue revealed significant downregulation in vivo. Additionally, the impact of heterologous expression of NR0B2 on cell cycle progression and proliferation in cells of renal origin was characterized. Monitoring fluorescence intensity of resazurin turnover in RCC-EW cells revealed no significant differences in metabolic activity in the presence of NR0B2. However, there was a significant decrease of cellular proliferation in cells overexpressing this NR, and NR0B2 was more efficient than currently used antiproliferative agents. Furthermore, flow cytometry analysis showed that heterologous overexpression of NR0B2 significantly reduced the amount of cells passing the G1 phase, while on the other hand, more cells in S/G2 phase were detected. Taken together, our data suggest that downregulation of NR0B2 may also play a role in renal cell carcinoma development and progression.

Differential genes in adipocytes induced from polycystic and non-polycystic ovary syndrome-derived human embryonic stem cells.

We explored the molecular mechanisms of obesity and insulin resistance in patients with polycystic ovary syndrome (PCOS) using a human embryonic stem cell model (hESCs). Three PCOS-derived and one non-PCOS-derived hESC lines were induced into adipocytes, and then total RNA was extracted. The differentially expressed PCOS-derived and non-PCOS-derived adipocytes genes were identified using the Boao Biological human V 2.0 whole genome oligonucleotide microarray. Signals of interest were then validated by real-time PCR. A total of 153 differential genes were expressed of which 91 genes were up-regulated and 62 down-regulated. Nuclear receptor subfamily 0, group B, member 2 (NR0B2) was an up-regulated gene, and the GeneChip CapitalBio® Molecule Annotation System V4.0 indicated that it was associated with obesity and diabetes (Ratio ≥ 2.0X). Multiple genes are involved in PCOS. Nuclear receptor subfamily 0, group B, member 2 may play a role in obesity and insulin resistance in patients with PCOS.

DAX1 regulatory networks unveil conserved and potentially new functions.

DAX1 is an orphan nuclear receptor with actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression from mammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish and mammalian DAX1 promoters share common sets of transcription factor frameworks which were also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordant with described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially new gene functions. The approach reported can be applied to genes from model and non-model species.