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To clarify the effect of retinoid X receptor-α/γ (RXR-α/γ) genes functional genetic variants (RXR-α rs4842194 G>A, RXR-γ rs100537 A>G and rs2134095 T>C) on the risk of gestational diabetes mellitus (GDM), a case-control study with 573 GDM patients and 740 pregnant women with normal glucose tolerance was performed in Guangxi area of China. An odds ratio (OR) with its corresponding 95% confidence interval (CI) was used to assess the strengths of the association between genetic variation and GDM. After adjustment of age and pre-BMI, the logistic regression analysis showed that the rs2134095 was significantly associated with GDM risk (CC vs. TT/TC: adjusted OR = 0.71, 95% CI = 0.56-0.90) in all subjects, and this result remained highly significant after Bonferroni's correction for multiple testing (P=0.004). The stratified analysis showed that rs2134095 was significantly associated with the risk of GDM among age > 30 years (adjusted OR = 0.61, 95% CI = 0.39-0.97), BMI > 22 kg/m2 (adjusted OR = 0.46, 95% CI = 0.30-0.70), systolic blood pressure (SBP) > 120 mmHg (adjusted OR = 1.96, 95% CI = 1.14-3.36), glycosylated hemoglobin A1c (HbA1c) < 6.5% (adjusted OR = 1.41, 95% CI = 1.11-1.78), TG ≤ 1.7 mmol/l (adjusted OR = 2.57, 95% CI = 1.45-4.53), TC ≤ 5.18 mmol/l (adjusted OR = 1.58, 95% CI = 1.13-2.22), high-density lipoprotein cholesterol (HDL-c) ≤ 1.5 mmol/l (adjusted OR = 1.70, 95% CI = 1.16-2.49) and low-density lipoprotein cholesterol (LDL-c) > 3.12 mmol/l (adjusted OR = 1.47, 95% CI = 1.08-2.00) subjects, under the recessive genetic model. We also found that rs2134095 interacted with age (Pinteraction=0.039), pre-BMI (Pinteraction=0.040) and TG (Pinteraction=0.025) influencing individual's genetic susceptibility to GDM. The rs2134095 T>C is significantly associated with the risk of GDM by effect of a single locus and/or complex joint gene-gene and gene-environment interactions. Larger sample-size and different population studies are required to confirm the findings., (© 2021 The Author(s).)

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL -/- ), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL -/- OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL -/- OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases., (©2021 The Authors. Stem Cells published by Wiley Periodicals LLC on behalf of AlphaMed Press 2021.)

Retinoid X receptors are members of the nuclear receptor family that regulate gene expression in response to retinoic acid and related ligands. Group 1 metabotropic glutamate receptors are G-protein coupled transmembrane receptors that activate intracellular signaling cascades in response to the neurotransmitter, glutamate. These two classes of molecules have been studied independently and found to play important roles in regulating neuronal physiology with potential clinical implications for disorders such as depression, schizophrenia, Parkinson's and Alzheimer's disease. Here we show that mice lacking the retinoid X receptor subunit, RXRγ, exhibit impairments in group 1 mGluR-mediated electrophysiological responses at hippocampal Schaffer collateral-CA1 pyramidal cell synapses, including impaired group 1 mGluR-dependent long-term synaptic depression (LTD), reduced group 1 mGluR-induced calcium release, and loss of group 1 mGluR-activated voltage-sensitive currents. These animals also exhibit impairments in a subset of group 1 mGluR-dependent behaviors, including motor performance, spatial object recognition, and prepulse inhibition. Together, these observations demonstrate convergence between the RXRγ and group 1 mGluR signaling pathways that may function to coordinate their regulation of neuronal activity. They also identify RXRγ as a potential target for the treatment of disorders in which group 1 mGluR signaling has been implicated.

Objective: To investigate the relationship between genetic polymorphisms of RXRG rs1467664, rs3753898 and the genetic susceptibility of type 2 diabetes in the Chinese Han population from South China., Methods: In our case-control study, single-nucleotide polymorphisms (SNPs) rs1467664 and rs3753898 were genotyped by SNPscanTM kit in 1092 patients with T2D as cases and 1092 normal persons as controls. The distributions of genotype and allele frequencies in two groups were analyzed by the SPSS 20.0 software., Results: The distribution of genotypes and alleles of RXRG rs3753898 was statistically significant between the two groups, but there was no significant difference in the distribution of genotypes and alleles of the rs1467664. Before and after the adjustment of age, sex and BMI, rs3753898 in the two groups had statistical significance under the additive, dominant and recessive models (P<0.05), but no statistical differences were found under the overdominance and co-dominant genetic models (P>0.05). There was no significant difference in the genetic models of rs1467664 between the two groups (P>0.05). The haplotype, which consists of rs1467664 allele T and rs3753898 allele A was a high-risk factor for T2D, OR=1.27, 95% CI (1.09-1.47), Padj=0.002., Conclusion: Our results showed that the single nucleotide polymorphism of RXRG rs3753898 may be related to genetic susceptibility of type 2 diabetes. The haplotype consisting of the allele T of rs1467664 and the allele A of rs3753898 is a risk factor for type 2 diabetes, suggesting that the genetic variation of RXRG gene may be the genetic cause of diabetes mellitus in the Chinese Han population., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Skeletal muscle, the largest part of the total body mass, influences energy and protein metabolism as well as maintaining homeostasis. Herein, we demonstrate that during murine muscle satellite cell and myoblast differentiation, transthyretin (TTR) can exocytose via exosomes and enter cells as TTR- thyroxine (T 4 ) complex, which consecutively induces the intracellular triiodothyronine (T 3 ) level, followed by T 3 secretion out of the cell through the exosomes. The decrease in T 3 with the TTR level in 26-week-old mouse muscle, compared to that in 16-week-old muscle, suggests an association of TTR with old muscle. Subsequent studies, including microarray analysis, demonstrated that T 3 -regulated genes, such as FNDC5 (Fibronectin type III domain containing 5, irisin) and RXRγ (Retinoid X receptor gamma), are influenced by TTR knockdown, implying that thyroid hormones and TTR coordinate with each other with respect to muscle growth and development. These results suggest that, in addition to utilizing T 4 , skeletal muscle also distributes generated T 3 to other tissues and has a vital role in sensing the intracellular T 4 level. Furthermore, the results of TTR function with T 4 in differentiation will be highly useful in the strategic development of novel therapeutics related to muscle homeostasis and regeneration., Competing Interests: The authors declare that they have no conflict of interest.

Background: Retinoid X Receptor Gamma (RXRG) is a member of the nuclear receptor superfamily and plays a role in tumour suppression. This study aims to explore the prognostic significance of RXRG in breast cancer., Methods: Primary breast cancer tissue microarrays (n = 923) were immuno-stained for RXRG protein and correlated with clinicopathological features, and patient outcome., Results: Nuclear RXRG expression was significantly associated with smaller tumour size (p = 0.036), lower grade (p < 0.001), lobular histology (p = 0.016), lower Nottingham Prognostic Index (p = 0.04) and longer breast cancer-specific survival (p < 0.001), and longer time to distant metastasis (p = 0.002). RXRG expression showed positive association with oestrogen receptor (ER)-related biomarkers: GATA3, FOXA1, STAT3 and MED7 (all p < 0.001) and a negative correlation with the Ki67 proliferation marker. Multivariate analysis demonstrated RXRG protein as an independent predictor of longer breast cancer-specific survival and distant metastasis-free survival. In the external validation cohorts, RXRG expression was associated with improved patients' outcome (p = 0.025). In ER-positive tumours, high expression of RXRG was associated with better patient outcome regardless of adjuvant systemic therapy. ER signalling pathway was the top predicted master regulator of RXRG protein expression (p = 0.005)., Conclusion: This study provides evidence for the prognostic value of RXRG in breast cancer particularly the ER-positive tumours.

This study was designed to investigate effects of xanthophylls on serum lipid profile (triglyceride, TG; cholesterol, CHO; high-density lipoprotein cholesterol, HDLC; and low-density lipoprotein cholesterol, LDLC) and nuclear factor (peroxisome proliferator-activated receptor gamma, PPARγ; PPAR gamma coactivator 1 alpha, PGC1α; retinoid X receptor gamma, RXRγ; and retinoic acid receptor alpha, RARα) gene expression of breeding hens and chicks. In experiment 1, 432 hens were divided into three groups and fed diets supplemented with 0 (as control group), 20 or 40 mg/kg xanthophylls. Blood was sampled at 7, 14, 21, 28 and 35 days of trial. Liver, duodenum, jejunum and ileum were sampled at 35 days of trial. Results showed that serum HDLC level of hens was increased after dietary 40 mg/kg xanthophyll addition for 21, 28 and 35 days, while serum TG, CHO and LDLC were not affected. Xanthophyll addition also increased PPARγ expression in jejunum, RXRγ expression in duodenum and jejunum, and RARα expression in liver and duodenum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg/kg xanthophyll diet of hens were fed diet containing either 0 or 40 mg/kg xanthophylls. Liver, duodenum, jejunum and ileum were sampled at 0, 7, 14 and 21 days after hatching. Blood samples were also collected at 21 days. Results showed that in ovo xanthophylls elevated PPARγ in duodenum and jejunum, and RXRγ and RARα in liver of chicks mainly within 1 week after hatching, while dietary xanthophylls increased serum HDLC level and PPARγ and RXRγ in liver from 2 weeks onwards. In conclusion, our research suggested xanthophylls can regulate serum lipid profile and nuclear factor expression in hens and chicks., (© 2017 Blackwell Verlag GmbH.)

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors., Methods: A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures., Results: Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina., Conclusions: In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Much evidence suggests an association between vitamin D deficiency and chronic diseases such as obesity and dyslipidemia. Although genetic factors play an important role in the etiology of these diseases, only a few studies have investigated the relationship between vitamin D-related genes and anthropometric and lipid profiles. The aim of this study was to investigate the association of three vitamin D-related genes with anthropometric and lipid parameters in 542 adult individuals. We analyzed the rs2228570 polymorphism in the vitamin D receptor gene (VDR), rs2134095 in the retinoid X receptor gamma gene (RXRG) and rs7041 in the vitamin D-binding protein gene (GC). Polymorphisms were genotyped by TaqMan allelic discrimination. Gene-gene interactions were evaluated by the general linear model. The functionality of the polymorphisms was investigated using the following predictors and databases: SIFT (Sorting Intolerant from Tolerant), PolyPhen-2 (Polymorphism Phenotyping v2) and Human Splicing Finder 3. We identified a significant effect of the interaction between RXRG (rs2134095) and GC (rs7041) on low-density lipoprotein cholesterol (LDL-c) levels (P=.005). Furthermore, our in silico analysis suggested a functional role for both variants in the regulation of the gene products. Our results suggest that the vitamin D-related genes RXRG and GC affect LDL-c levels. These findings are in agreement with other studies that consistently associate vitamin D and lipid profile. Together, our results corroborate the idea that analyzing gene-gene interaction would be helpful to clarify the genetic component of lipid profile., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Vitamin D (VD) deficiency during pregnancy has been repeatedly linked to an increased gestational diabetes mellitus (GDM) risk. We sought to determine the influences of genetic variants in vitamin D signaling pathways on the risk of GDM. In this study, we genotyped 15 single nucleotide polymorphisms (SNPs) within 8 representative genes (CYP27A1, CYP27B1, CYP24A1, VDR, RXRA, RXRB, RXRG and GC) of the vitamin D signaling pathways in a case-control study with 964 GDM cases and 1,021 controls using the Sequenom MassARRAY iPLEX platform. Logistic regression analyses in additive model showed that GC rs16847024 C>T, RXRG rs17429130 G>C and RXRA rs4917356 T>C were significantly associated with the increased risk of GDM (adjusted OR = 1.31, 95% CI = 1.10-1.58 for rs16847024; adjusted OR = 1.28, 95% CI = 1.04-1.57 for rs17429130; adjusted OR = 1.28, 95% CI = 1.06-1.54 for rs4917356). And GDM risk significantly increased with the increasing number of variant alleles of the three SNPs in a dose-dependent manner (P for trend < 0.001). Moreover, the combined effect of the three SNPs on GDM occurrence was more prominent in older women (age > 30). Further interactive analyses also detected a significantly multiplicative interaction between the combined variant alleles and age on GDM risk (P = 0.035). Together, these findings indicate that GC rs16847024, RXRG rs17429130 and RXRA rs4917356 were candidate susceptibility markers for GDM in Chinese females. Further validation studies with different ethnic background and biological function analyses were needed.

Familial isolated pituitary adenoma (FIPA) is a rare genetic disorder. In a subset of FIPA families AIP germline mutations have been reported, but in most FIPA cases the exact genetic defect remains unknown. The present study aimed to determine the genetic basis of FIPA in a Brazilian family. Three siblings presented with isolated prolactin genes. Further mutation screening was performed using whole-exome sequencing and all likely causative mutations were validated by Sanger sequencing. In silico analysis and secreting pituitary adenoma diagnosed through clinical, biochemical and imaging testing. Sanger sequencing was used to genotype candidate prolactinoma-mutated additional predictive algorithms were applied to prioritize likely pathogenic variants. No mutations in the coding and flanking intronic regions in the MEN1, AIP and PRLR genes were detected. Whole-exome sequencing of three affected siblings revealed novel, predicted damaging, heterozygous variants in three different genes: RXRG, REXO4 and TH. In conclusion, the RXRG and TH possibly pathogenic variants may be associated with isolated prolactinoma in the studied family. The possible contribution of these genes to additional FIPA families should be explored., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Retinoids have been implicated as pharmacological agents for the prevention and treatment of various types of cancers, including breast cancers. We analyzed 27 newly synthesized retinoids for their bioactivity on breast, liver, and colon cancer cells. Majority of the retinoids demonstrated selective bioactivity on breast cancer cells. Retinoid 17 had a significant inhibitory activity (IC50 3.5 μM) only on breast cancer cells while no growth inhibition observed with liver and colon cancer cells. The breast cancer selective growth inhibitory action by retinoid 17 was defined as p21-dependent cell death, reminiscent of senescence, which is an indicator of targeted receptor mediated bioactivity. A comparative analysis of retinoid receptor gene expression levels in different breast cancer cells and IC50 values of 17 indicated the involvement of Retinoid X receptors in the cytotoxic bioactivity of retinoid 17 in the senescence associated cell death. Furthermore, siRNA knockdown studies with RXRγ induced decrease in cell proliferation. Therefore, we suggest that retinoid derivatives that target RXRγ, can be considered for breast cancer therapies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases.

Virus-induced exacerbations often lead to further impairment of lung function in chronic obstructive pulmonary disease. IL-15 is critical in antiviral immune responses. Retinoic acid (RA) signaling plays an important role in tissue maintenance and repair, particularly in the lung. We studied RA signaling and its relation to IL-15 in the lung during cigarette smoke (CS) exposure and influenza virus infection. In vivo studies show that RA signaling is diminished by long-term CS exposure or influenza virus infection alone, which is further attenuated during infection after CS exposure. RA receptor β (RARβ) is specifically decreased in the lung of IL-15 transgenic (overexpression; IL-15Tg) mice, and a greater reduction in RARβ is found in these mice compared with wild-type (WT) mice after infection. RARβ is increased in IL-15 knockout (IL-15KO) mice compared with WT mice after infection, and the additive effect of CS and virus on RARβ down-regulation is diminished in IL-15KO mice. IL-15 receptor α (IL-15Rα) is increased and RARβ is significantly decreased in lung interstitial macrophages from IL-15Tg mice compared with WT mice. In vitro studies show that IL-15 down-regulates RARβ in macrophages via IL-15Rα signaling during influenza virus infection. These studies suggest that RA signaling is significantly diminished in the lung by CS exposure and influenza virus infection. IL-15 specifically down-regulates RARβ expression, and RARβ may play a protective role in lung injury caused by CS exposure and viral infections.

Purpose: Resolution of aberrant epigenetic changes leading to altered gene expression during transformation and tumor progression is pertinent for mechanistic understanding of disrupted pathways in cancer. Such changes provide for biomarkers that can be applied in drug screening and improved disease management., Experimental Design: Genome-wide profiling and analyses of promoter DNA methylation, histone modifications, and gene expression of an in vitro progression model of serous ovarian adenocarcinoma were carried out. Similar in silico analyses and comparison of methylation and gene expression of early- and late-grade ovarian cancer samples in The Cancer Genome Atlas assigned a clinical relevance to our study. Candidate biomarkers were evaluated for epigenetic drug treatments in experimental animal models on a background of differing tumor cell responses arising from intratumor heterogeneity., Results: Differentially regulated genes during tumor progression were identified through the previously mentioned analyses as candidate biomarkers. In examining the tumor suppressor PTGIS as a potential biomarker for treatment with either 5-Aza-dC or TSA, 5-Aza-dC effectively stabilized cell cycling, restricted genetic instability, and derepressed PTGIS expression, while TSA led to emergence of drug-resistant progenitors lacking PTGIS expression. Profiling MEST and RXRγ for curcumin and CBB1007, respectively, indicated an inability of curcumin and CBB1007 in restricting residual tumor regenerative capabilities., Conclusions: Our study provides novel insights into epigenetic regulation in ovarian cancer progression and potential biomarkers for evaluating efficacy of epigenetic drugs in restricting residual tumor regeneration. Such approaches may assign a new functional interpretation of drug efficacy and cell tumor responses in ovarian cancer., (©2015 American Association for Cancer Research.)

Retinoid X receptors (RXRs) play a role as master regulators because of their capacity to form heterodimers with other nuclear receptors (NRs). Accordingly, retinoid signaling is involved in multiple biologic processes, including development, cell differentiation, metabolism, and cell death. However, the role and function of RXRs in different heterodimer complexes remain unidentified, mainly because most RXR drugs (called rexinoids) are not selective of specific heterodimer complexes. The lack of selectivity strongly limits the use of rexinoids for specific therapeutic approaches. To better characterize rexinoids at specific NR complexes, we have developed and optimized luciferase (Luc) protein complementation(PCA)-based bioluminescence resonance energy transfer (BRET) assays that can directly measure recruitment of a coactivator (CoA) motif fused to yellow fluorescent protein (YFP) by specific NR dimers. To validate the assays, we compared rexinoid modulation of CoA recruitment by the RXR homodimer and by the heterodimers Nur77/RXR and Nurr1/RXR. Results revealed that some rexinoids display selective CoA recruitment activities with homo- or heterodimer complexes. In particular, SR11237 (BMS649) has stronger potency for recruitment of CoA motif and transcriptional activity with the heterodimer Nur77/RXR than other complexes. This technology should be useful in identifying new compounds with specificity for individual dimeric species formed by NRs., (© FASEB.)

Key Points: Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation., Abstract: Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, β and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Cocaine abuse represents an immense societal health and economic burden for which no effective treatment currently exists. Among the numerous intracellular signaling cascades impacted by exposure to cocaine, increased and aberrant production of pro-inflammatory cytokines in the CNS has been observed. Additionally, we have previously reported a decrease in retinoid-X-receptor-gamma (RXR-γ) in brains of mice chronically exposed to cocaine. Through obligate heterodimerization with a number of nuclear receptors, RXRs serve as master regulatory transcription factors, which can potentiate or suppress expression of a wide spectrum of genes. Little is known about the regulation of RXR levels, but previous studies indicate cellular stressors such as cytokines negatively regulate levels of RXRs in vitro. To evaluate the mechanism underlying the cocaine-induced decreases in RXR-γ levels observed in vivo, we exposed neurons to cocaine in vitro and examined pathways which may contribute to disruption in RXR signaling, including activation of stress pathways by cytokine induction. In these studies, we provide the first evidence that cocaine exposure disrupts neuronal RXR-γ signaling in vitro by promoting its nuclear export and degradation. Furthermore, we demonstrate this effect may be mediated, at least in part, by cocaine-induced production of TNF-α and its downstream effector c-Jun-NH-terminal kinase (JNK). Findings from this study are therefore applicable to both cocaine abuse and to pathological conditions characterized by neuroinflammatory factors, such as neurodegenerative disease.

Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting-feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting-feeding cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To characterize brown adipose tissue (BAT) in the human perirenal adipose tissue depot., Method: Perirenal adipose tissue biopsies were obtained from 55 healthy kidney donors. Expression analysis was performed using microarray, real-time PCR, immunoblotting and immunohistochemistry. Additional studies using human stem cells were performed., Results: UCP1 gene expression analysis revealed a large intra-individual variation in the perirenal adipose tissue biopsies. Both multi- and unilocular UCP1-positive adipocytes were detected in several of the adipose tissue samples analyzed by immunohistochemical staining. Microarray analysis identified 54 genes that were overexpressed in UCP1-positive perirenal adipose tissue. Real-time PCR analysis of BAT candidate genes revealed a set of genes that were highly correlated to UCP1 and a set of three transcription factor genes (PRDM16, PGC1α, and RXRγ) that were highly correlated to each other. RXRγ displayed nuclear immunoreactivity in brown adipocytes and an increased gene expression during brown adipogenesis in human stem cells., Conclusion: Our data provides the first molecular characterization of BAT in the perirenal adipose tissue depot. Furthermore, it highlights the transcription factor RXRγ as a new player in BAT development., (Copyright © 2014 The Obesity Society.)

The OSCAR test, a clinical device that uses counterphase flicker photometry, is believed to be sensitive to the relative numbers of long-wavelength and middle-wavelength cones in the retina, as well as to individual variations in the spectral positions of the photopigments. As part of a population study of individual variations in perception, we obtained OSCAR settings from 1058 participants. We report the distribution characteristics for this cohort. A randomly selected subset of participants was tested twice at an interval of at least one week: the test-retest reliability (Spearman's rho) was 0.80. In a whole-genome association analysis we found a provisional association with a single nucleotide polymorphism (rs16844995). This marker is close to the gene RXRG, which encodes a nuclear receptor, retinoid X receptor γ. This nuclear receptor is already known to have a role in the differentiation of cones during the development of the eye, and we suggest that polymorphisms in or close to RXRG influence the relative probability with which long-wave and middle-wave opsin genes are expressed in human cones.

Objectives: Alterations in enzymatic antioxidant defense systems lead to a deficit of cognitive functions and altered hippocampal synaptic plasticity. The objectives of this study were to investigate endogenous rhythms of catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as CREB1 mRNA, in the rat hippocampus, and to evaluate to which extent the vitamin A deficiency could affect those temporal patterns., Methods: Rats from control and vitamin A-deficient (VAD) groups received a diet containing 4000 IU of vitamin A/kg diet, or the same diet devoid of vitamin A, respectively, during 3 months. Rats were maintained under 12-hour-dark conditions, during 10 days before the sacrifice. Circadian rhythms of CAT, GPx, RXRγ, and CREB1 mRNA levels were determined by reverse transcriptrase polymerase chain reaction in hippocampus samples isolated every 4 hours during a 24-hour period. CAT and GPx enzymatic activities were also determined by kinetic assays. Regulatory regions of clock and antioxidant enzymes genes were scanned for E-box, RXRE, and CRE sites., Results: E-box, RXRE, and CRE sites were found on regulatory regions of GPx and CAT genes, which display a circadian expression in the rat hippocampus. VAD phase shifted CAT, GPx, and RXRγ endogenous rhythms without affecting circadian expression of CREB1., Discussion: CAT and GPx expression and enzymatic activity are circadian in the rat hippocampus. The VAD affected the temporal patterns antioxidant genes expression, probably by altering circadian rhythms of its RXR receptors and clock factors; thus, it would impair the temporal orchestration of hippocampal daily cognitive performance.

Tumor metastasis and lack of NKG2D ligand (NKG2DL) expression are associated with poor prognosis in patients with colon cancer. Here, we found that spironolactone (SPIR), an FDA-approved diuretic drug with a long-term safety profile, can up-regulate NKG2DL expression in multiple colon cancer cell lines by activating the ATM-Chk2-mediated checkpoint pathway, which in turn enhances tumor elimination by natural killer cells. SPIR can also up-regulate the expression of metastasis-suppressor genes TIMP2 and TIMP3, thereby reducing tumor cell invasiveness. Although SPIR is an aldosterone antagonist, its antitumor effects are independent of the mineralocorticoid receptor pathway. By screening the human nuclear hormone receptor siRNA library, we identified retinoid X receptor γ (RXRγ) instead as being indispensable for the antitumor functions of SPIR. Collectively, our results strongly support the use of SPIR or other RXRγ agonists with minimal side effects for colon cancer prevention and therapy.

HIV lipodystrophy (HIVLD), associated with combination antiretroviral therapy (cART), leads to metabolic and cardiovascular diseases. Nuclear receptors play a central role in lipid homoeostasis and drug disposition; their genetic variants may predispose an individual to the development of HIVLD. DNA samples obtained from cART-treated HIV-positive patients with (HIVLD+; 124) and without (HIVLD-; 56) HIVLD were genotyped for 77 single nucleotide polymorphisms in nine nuclear receptor genes. Statistical analysis was carried out using Haploview software and by logistic regression. Three single nucleotide polymorphisms in RXRγ (rs2134095, rs113471, rs2194899) and its haplotypes (HIVLD+, 54%; HIVLD-, 40.6%; P=0.02) showed significant association with HIVLD. Multivariate analysis identified time since diagnosis (P=0.001) and carriage of the RXRγ haplotype (P=0.02) to be independently associated with HIVLD. Genetic variation in RXRγ, a common binding partner of nuclear receptors that modulate lipid homoeostasis and drug disposition, may contribute to the development of HIVLD in cART-treated HIV patients. These results need replication in other cohorts.

Purpose: Retinoic acid (RA) treatment has been used for redifferentiation of metastatic thyroid cancer with loss of radioiodine uptake. The aim of this study was to improve the understanding of RA resistance and investigate the role of bexarotene in thyroid cancer cells., Experimental Design: A model of thyroid cancer cell lines with differential response to RA was used to evaluate the biological effects of retinoid and rexinoid and to correlate this with RA receptor levels. Subsequently, thyroid cancer patients were treated with 13-cis RA and bexarotene and response evaluated on radioiodine uptake reinduction on posttherapy scan and conventional imaging., Results: In thyroid cancer patients, 13-cis RA resistance can be bypassed in some tumors by bexarotene. A decreased tumor growth without differentiation was observed confirming our in vitro data. Indeed, we show that ligands of RARs or RXRs exert different effects in thyroid cancer cell lines through either differentiation or inhibition of cell growth and invasion. These effects are associated with restoration of RARβ and RXRγ levels and downregulation of NF-κB targets genes. We show that bexarotene inhibits the transactivation potential of NF-κB in an RXR-dependent manner through decreased promoter permissiveness without interfering with NF-κB nuclear translocation and binding to its responsive elements. Inhibition of transcription results from the release of p300 coactivator from NF-κB target gene promoters and subsequent histone deacetylation., Conclusion: This study highlights dual mechanisms by which retinoids and rexinoids may target cell tumorigenicity, not only via RARs and RXRs, as expected, but also via NF-κB pathway., (©2011 AACR.)

TDP-43 is a nuclear protein implicated in the pathogenesis of several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration, with broad involvement in numerous stages of RNA processing ranging from transcription to translation. In diseased neurons, TDP-43 mostly aggregates in the cytoplasm, suggesting that a loss of protein function in the nucleus may play an important role in neurodegeneration. A better understanding of TDP-43 general nuclear functions is therefore an essential step to evaluate this possibility. Presently, the TDP-43 best-characterized functional property is its ability to modulate pre-mRNA splicing when binding in proximity of 3'SS acceptor sequences. In this work, using a variety of artificial and natural splicing substrates, we have investigated the effects of TDP-43 binding to UG repeats in the vicinity of 5'SS donor sequences. In general, our results show that UG repeats are not powerful splicing regulatory elements when located near to exonic 5'SS sequences. However, in cases like the BRCA1, ETF1, and RXRG genes, TDP-43 binding to natural UG-repeated sequences can act as either an activator or a suppressor of 5'SS recognition, depending on splice site strength and on the presence of additional splicing regulatory sequences. The results of this analysis suggest that a role of UG repeats/TDP-43 in 5'SS recognition may exists and may become critical in the presence of mutations that weaken the 5'SS. The general rule that can be drawn at the moment is that the importance of UG repeats near 5' splice sites should always be experimentally validated on a case-by-case basis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Retinoid-X receptor (RXR) is one of the members of the nuclear hormone receptor superfamily. It forms heterodimers with many nuclear receptors, such as the peroxisome proliferative-activated receptor, which has been proposed to be involved in diabetic complications, including retinopathy. A recent study revealed that RXR-alpha has antioxidant properties and is associated with diabetic retinopathy. We found that the RXR-gamma gene is involved in the pathogenesis of diabetic nephropathy. We also hypothesized that the RXR-gamma gene has a role in the development of diabetic retinopathy. We examined 213 diabetic patients, who were divided into retinopathy or no retinopathy groups. Nine selected single nucleotide polymorphisms (SNPs) in the RXR-gamma gene were evaluated. The diabetic retinopathy group had longer diabetes duration, higher body mass indexes, and higher systolic blood pressure, as well as higher concentrations of fasting plasma glucose, blood urine nitrogen, and creatine. One SNP--rs3818569 of the RXR-gamma gene was found to be associated with increased risk for diabetic retinopathy in both allele and genotype frequencies (P = 0.0023 and 0.0057, respectively). Analysis with multivariate logistic regression revealed that the dominant RXR-gamma GG genotype is a risk factors for the development of diabetic retinopathy (odds ratio = 2.388; 95% confidence interval = 1.17-4.875). We conclude that the RXR-gamma rs3818569 SNP is associated with diabetic retinopathy development in the Taiwanese population.

Background: Di-n-butyl phthalate (DBP), a chemical widely used in many consumer products, is estrogenic and capable of producing seriously reproductive and developmental effects in laboratory animals. However, recent in vitro studies have shown that DBP and mono-n-butyl phthalate (MBP), the major metabolite of DBP, possessed thyroid hormone receptor (TR) antagonist activity. It is therefore important to consider DBP and MBP that may interfere with thyroid hormone system., Methodology/principal Findings: Nieuwkoop and Faber stage 51 Xenopus laevis were exposed to DBP and MBP (2, 10 or 15 mg/L) separately for 21 days. The two test chemicals decelerated spontaneous metamorphosis in X. laevis at concentrations of 10 and 15 mg/L. Moreover, MBP seemed to possess stronger activity. The effects of DBP and MBP on inducing changes of expression of selected thyroid hormone response genes: thyroid hormone receptor-beta (TRβ), retinoid X receptor gamma (RXRγ), alpha and beta subunits of thyroid-stimulating hormone (TSHα and TSHβ) were detected by qPCR at all concentrations of the compounds. Using mammalian two-hybrid assay in vitro, we found that DBP and MBP enhanced the interactions between co-repressor SMRT (silencing mediator for retinoid and thyroid hormone receptors) and TR in a dose-dependent manner, and MBP displayed more markedly. In addition, MBP at low concentrations (2 and 10 mg/L) caused aberrant methylation of TRβ in head tissue., Conclusions: The current findings highlight potential disruption of thyroid signalling by DBP and MBP and provide data for human risk assessment.

Background: Omega-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), have antidepressant and promnemonic functions. The mechanisms of such activities are still elusive and may involve retinoid X receptors (RXRs), transcription factors known to bind DHA in vitro., Methods: Promnemonic and antidespair activities of acute DHA treatment were tested in BALBcByJ mice using spontaneous alternation and forced swim test, respectively. The involvement of retinoid receptors in such DHA activities was investigated using RXR and/or retinoic acid receptor (RAR) agonists to mimic DHA activities or a synthetic pan-RXR antagonist to block them. Involvement of RXR isotypes was analyzed using the same tasks and delayed nonmatch to place for working memory in RXRγ knockout mice., Results: Docosahexaenoic acid decreased despair behavior and improved working memory in BALBcByJ mice. Such effects were suppressed by co-treatment with BR1211, a pan-RXR antagonist, whereas a pan-RXR agonist, UVI2108, mimicked DHA activities. Retinoic acid (RA), a natural ligand of RXRs, also reduced despair behavior and improved working memory and such activities did not require activation of RARs, as RA effects were abolished by co-treatment with BR1211 and they were not reproduced by TTNPB, a pan-RAR agonist. The RXRγ knockout mice displayed increased despair and deficits in working memory, which were insensitive to DHA and pan-RXR agonist treatments, whereas DHA or UVI2108 reversed these deficits in RXRγ heterozygous mice., Conclusions: Our data suggest that RXRs are a converging point in mediating DHA and RA modulations of despair behavior and working memory and that RXRγ is the predominant RXR isotype in these regulations., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The expression of retinoid X receptor γ (RXRγ) and the clinicopathological parameters of total 69 patients with papillary thyroid carcinoma (PTC) larger than 1 cm were examined. The PTCs were classified into two groups according to the presence of loss of cellular polarity/cohesiveness (LOP/C). The expression of RXRγ mRNA was examined by reverse transcriptase polymerase chain reaction and quantitative real-time PCR. The RXRγ mRNA up-regulation was found to be positively correlated with extrathyroid invasion (r = 0.293, P = 0.019), advanced tumor stage (r = 0.318, P = 0.016) and lymph node metastasis (LNM) (r = 0.338, P = 0.005), as well as LOP/C (r = 0.345, P = 0.004), which was proposed as a histological characteristic of poor cellular differentiation. The RXRγ mRNA expression, as well as extrathyroid invasion, LOP/C and advanced tumor stage, was further confirmed to be one of the independent predictive factors (Odds ratio: 6.545; 95% confidence interval: 1.575-27.208) of LNM using multivariate analysis. These results suggest that RXRγ may play a role in the dedifferentiation and metastasis of PTC., (© 2010 The Authors. Pathology International © 2010 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Background: Schwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type., Methodology/principal Findings: In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-β mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability., Conclusions/significance: All together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology.

Background: Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism., Methodology/principal Findings: RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs., Conclusions/significance: Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

We aimed to identify which cytochrome P-450 (CYP) family/subfamily, as well as related transcription factor(s), is responsible for the estrogen-dependent synthesis of epoxyeicosatrienoic acids (EETs) to initiate shear stress-induced vasodilation. Microarray analysis indicated a significant upregulation of CYP2C29 and retinoid X receptor gamma (RXRgamma) in isolated mesenteric arteries/arterioles of female endothelial nitric oxide synthase-knockout mice, a result that was validated by real-time RT-PCR. The cannulated vessels were then perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress dose-dependently stimulated the release of EETs into the perfusate, associated with an EET-mediated vasodilation, in which predominantly 14,15-EET and 11,12-EET contributed to the responses ( approximately 87.4% of total EETs). Transfection of vessels with CYP2C29 siRNA eliminated the release of EETs into the perfusate, which was evidenced by an abolished vasodilation, and confirmed by RT-PCR and Western blot analyses. Knockdown of RXRgamma in these vessels significantly inhibited the production of EETs, parallel to a reduced vasodilation. RXRgamma siRNA not only silenced the vascular RXRgamma expression, but synchronously downregulated CYP2C29 expression, leading to a reduced EET synthesis. In conclusion, our data provide the first evidence for a specific signaling cascade, by which estrogen potentially activates the CYP2C29 gene in the absence of nitric oxide, to synthesize EETs in response to shear stress, via an RXRgamma-related regulatory mechanism.

Objective: Smoking decreases insulin action and increases the risk of type 2 diabetes in humans. Mechanisms responsible for smoking-induced insulin resistance are unclear. We hypothesized smokers would have increased intramuscular triglyceride (IMTG) and diacylglycerol (DAG) concentration and decreased fractional synthesis rate (FSR) compared with nonsmokers., Research Design and Methods: Nonsmokers (n = 18, aged 20 +/- 0.5 years, BMI 22 +/- 0.4 kg/m(2), body fat 20 +/- 2%, 0 cigarettes per day) and smokers (n = 14, aged 21 +/- 0.7 years, BMI 23 +/- 0.4 kg/m(2), body fat 20 +/- 3%, 18 +/- 0.7 cigarettes per day) were studied in a fasted condition after a standardized diet. [U-(13)C]palmitate was infused during 4 h of rest followed by a skeletal muscle biopsy and intravenous glucose tolerance test., Results: Smokers were less insulin sensitive (S(i)) compared with nonsmokers (S(i) 5.28 +/- 0.5 nonsmokers vs. 3.74 +/- 0.3 smokers 10(-4) x microU(-1) x ml(-1), P = 0.03). There were no differences in IMTG or DAG concentration (IMTG 24.2 +/- 3.4 nonsmokers vs. 27.2 +/- 5.9 smokers microg/mg dry wt, DAG 0.34 +/- 0.02 nonsmokers vs. 0.35 +/- 0.02 smokers microg/mg dry wt) or IMTG FSR between groups (0.66 +/- 0.1 nonsmokers vs. 0.55 +/- 0.09 smokers %/hr). Intramuscular lipid composition was different, with increased percent saturation of IMTG (32.1 +/- 1.2 nonsmokers vs. 35.2 +/- 1.0 smokers %, P = 0.05) and DAG (52.8 +/- 1.7 nonsmokers vs. 58.8 +/- 2.2 smokers %, P = 0.04) in smokers. Smokers had significantly decreased peroxisome proliferator-activated receptor-gamma (1.76 +/- 0.1 nonsmokers vs. 1.42 +/- 0.11 smokers arbitrary units [AU], P = 0.03) and increased monocyte chemotactic protein-1 (3.11 +/- 0.41 nonsmokers vs. 4.83 +/- 0.54 smokers AU, P = 0.02) mRNA expression compared with nonsmokers. We also found increased insulin receptor substrate-1 Ser(636) phosphorylation in smokers compared with nonsmokers (0.73 +/- 0.08 nonsmokers vs. 1.14 +/- 0.09 smokers AU, P = 0.002)., Conclusions: These data suggest: 1) IMTG concentration and turnover are not related to alterations in insulin action in smokers compared to nonsmokers, 2) increased saturation of IMTG and DAG in skeletal muscle may be related to insulin action, and 3) basal inhibition of insulin receptor substrate-1 may decrease insulin action in smokers.

The nuclear hormone receptors (NHRs), a family of transcription factors, bind directly to the hormone response elements (HREs) to regulate gene expression. In this study, we describe a comprehensive NHR-HRE profiling analysis with a new high-throughput DNA binding assay system utilizing wheat germ cell-free protein production and fluorescence correlation spectroscopy (FCS). This approach revealed NHR binding to natural response elements and new heterodimeric NHR-HRE bindings. We analyzed 408 possible binding combinations between 34 human NHRs and 12 different HREs, and identified 205 NHR-HRE binding combinations, 124 of which have not been previously reported. Thus, this study provides a novel biochemical classification of the human NHRs, as well as describing a novel approach to the large-scale analysis of DNA-protein interactions.

Type 2 diabetes (T2D) has been linked to chromosome 1q21-24 in multiple samples, including a Utah family sample. Variants in 13 of the numerous candidate genes in the 1q region were tested for association with T2D in a Utah case-control sample. The most promising, 19 variants in 6 candidates, were genotyped on the Utah family sample. Herein, we tested the 19 variants individually and in pairs for an effect on T2D risk in family members using a logistic regression model that accounted for gender, age, and BMI and attributed residual genetic effects to a polygenic component. Seven variants increased risk significantly through 5 pairs of interactions. The significant variant pairs were apolipoprotein A-II (APOA2) rs6413453 interacting with calsequestrin 1 (CASQ1) rs617698, dual specificity phosphatase 12 (DUSP12) rs1503814, and retinoid X receptor gamma (RXRG) rs10918169, a poly-T insertion-deletion polymorphism in liver pyruvate kinase (PKLR) interacting with APOA2 rs12143180, and DUSP12 rs1027702 interacting with RXRG rs10918169. Genotypes of these 5 variant pairs accounted for 25.8% of the genetic variance in T2D in these pedigrees.

Retinoic X receptor-gamma (RXRG) gene was studied as a candidate gene for the twinning trait of bovine. A new SNP A1941G was detected by sequencing at 3'UTR. Different genotypes were determined in Luxi monotocous cows, Luxi twinning cows, Chinese Simmental cows, Angus cows and Simmental x Mongolia cows by restriction fragment length polymorphism (RFLP). The value of polymorphism information content indicated that this was a moderate polymorphism in Luxi monotocous cows and Luxi twinning cows. The chi(2) test indicated that the polymorphic locus in Luxi twinning cows did not fit Hardy-Weinberg equilibrium (Plt;0.05). The chi(2) test of associational analysis between genotypic distribution and twinning or monotocous trait in Luxi cows showed that the difference was very significant (Plt;0.01).

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has essential roles in glucose homeostasis, and thiazolidinedione PPARgamma agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARgamma and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARgamma inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet alpha-cell line, ligand-bound PPARgamma was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARgamma ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARgamma-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARgamma-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARgamma DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARgamma is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARgamma-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARgamma-RXR.

Purpose: The active form of vitamin D3, that is 1alpha,25-dihydroxyvitamin D3, binds with vitamin D receptor, which forms a complex with retinoid X receptors alpha, beta and gamma to manifest antitumor effects. We examined the expression of vitamin D receptor and retinoid X receptors in renal cell carcinoma and elucidated the prognostic significance of these receptors., Materials and Methods: We performed immunohistochemical examination of vitamin D receptor, and retinoid X receptors alpha, beta and gamma in nephrectomized specimens of 68 patients with renal cell carcinoma. We analyzed the correlation between the expression of these receptors and clinicopathological parameters or patient survival. Mean followup was 68.2 months., Results: No significant correlation was found between the expression of vitamin D receptor, retinoid X receptor alpha or beta and clinicopathological parameters. In contrast, retinoid X receptor gamma expression correlated significantly with tumor stage (p = 0.009) and distant metastasis (p = 0.005). The 5-year cancer specific survival rate was higher in patients with retinoid X receptor gamma positive renal cell carcinoma than those with retinoid X receptor gamma negative renal cell carcinoma (79.3% vs 40.0%, p <0.05). Cox regression analysis revealed that retinoid X receptor gamma expression, tumor status and lymph node status were significant independent prognostic factors in patients with renal cell carcinoma (p <0.05). A significant correlation was observed between the expression of retinoid X receptor gamma and tumor stage, distant metastasis or the 5-year cancer specific survival rate. Furthermore, retinoid X receptor gamma expression was an independent prognostic factor in patients with renal cell carcinoma., Conclusions: Our observations suggest that alterations of vitamin D receptor and retinoid X receptor expression may be involved in renal carcinogenesis and retinoid X receptor gamma expression may be a useful prognostic marker in patients with renal cell carcinoma.

Objective: To apply linkage disequilibrium (LD) maps to associations studies with high throughput single nucleotide polymorphisms (SNPs)., Methods: Seven hundred and fifty-four SNPs were genotyped in 160 Shanghai Chinese. LD maps were constructed in cases and controls separately. By comparing the decline of LD unit with distance between the two groups, disease susceptible loci were estimated. This method was compared with traditional analyses including LD analysis, single SNP and haplotype analyses., Results: The analysis of LD maps could detect the chromosome regions with different LD patterns between the cases and controls. The alleles and/or haplotypes frequencies of SNPs within the regions had significantly different distributions or trends of significantly different distributions., Conclusion: This method may be applied to analyze the data from association studies with high throughput SNPs genotype information.

Objective: The genetic background of familial combined hyperlipidemia (FCHL) has not been fully clarified. Because several nuclear receptors play pivotal roles in lipid metabolism, we tested the hypothesis that genetic variants of nuclear receptors contribute to FCHL., Methods and Results: We screened all the coding regions of the PPARalpha, PPARgamma2, PPARdelta, FXR, LXRalpha, and RXRgamma genes in 180 hyperlipidemic patients including 60 FCHL probands. Clinical characteristics of the identified variants were evaluated in other 175 patients suspected of coronary disease. We identified PPARalpha Asp140Asn and Gly395Glu, PPARgamma2 Pro12Ala, RXRgamma Gly14Ser, and FXR -1g->t variants. Only RXRgamma Ser14 was more frequent in FCHL (15%, P<0.05) than in other primary hyperlipidemia (4%) and in controls (5%). Among patients suspected of coronary disease, we identified 9 RXRgamma Ser14 carriers, who showed increased triglycerides (1.62+/-0.82 versus 1.91+/-0.42 [mean+/-SD] mmol/L, P<0.05), decreased HDL-cholesterol (1.32+/-0.41 versus 1.04+/-0.26, P<0.05), and decreased post-heparin plasma lipoprotein lipase protein levels (222+/-85 versus 149+/-38 ng/mL, P<0.01). In vitro, RXRgamma Ser14 showed significantly stronger repression of the lipoprotein lipase promoter than RXRgamma Gly14., Conclusion: These findings suggest that RXRgamma contributes to the genetic background of FCHL.

Because of the retinoic acid receptor-alpha (RARalpha) gene's involvement in acute promyelocytic leukemia, the important role of RARs in hematopoiesis is now well established. However, relatively few studies of hematopoiesis have focused on the role of the retinoid X receptors (RXRs), the obligate heterodimeric partners of the RARs. We sought to establish whether conditional targeting of RXRalpha in early hematopoietic progenitors, ideally to the level of the hematopoietic stem cell (HSC), would compromise hematopoiesis. For hematopoietic targeting of RXRalpha, we characterized IFN-inducible MxCre mice for use in studying the role of RXRalpha in hematopoiesis. We established that MxCre executes recombination of loxP-flanked RXRalpha in hematopoietic progenitors immunophenotypically enriched for HSC, leading to widespread and sustained targeting of RXRalpha in hematopoietic cells. However, we found no evidence of hematologic compromise in mice lacking RXRalpha, suggesting that RXRalpha is dispensable for normal murine hematopoiesis. Nonetheless, RXRalpha null bone marrow cells cultured in methylcellulose form colonies more efficiently than bone marrow cells obtained from control mice. This result suggests that although RXRalpha is not required for murine hematopoiesis, there may be hematopoietic signaling pathways that respond selectively to RXRalpha or settings in which combined expression of RXR (alpha, beta, and gamma) is limiting.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes, accounting for the majority of patients with end-stage renal disease. The molecular pathogenesis of DN involves multiple pathways in a complex, partially resolved manner. The paper presents an exploratory epistatic study for DN. Association analysis were performed on 231 SNP loci in a cohort of 264 type 2 diabetes patients, followed by the epistasis analysis using the multifactor dimensionality reduction and the genetic algorithm with Boolean algebra. A two-locus epistatic effect of EGFR and RXRG was identified, with a cross-validation consistency of 91.7%.

Butyrate induces apoptosis of various cancer cell lines in a p53-independent manner and inhibits the proliferation of cancer cells. In a previous report, we reported a significant reduction in tumor incidence in rat colon as a result of dietary sodium gluconate (GNA). The stimulation of apoptosis through enhanced butyrate production in the large intestine was involved in the antitumorigenic effect of GNA. In the present study, a cDNA microarray analysis was performed to investigate the particular mechanism involved in the antitumorigenic effect of GNA. Some up-regulated genes suggested by microarray analysis were further evaluated using real-time PCR. A microarray revealed that GNA regulates the expression of retinoic acid receptor (RAR) and retinoid X receptor (RXR), and several genes known as the target of retinoids in cancer cells. In other words, the antitumorigenic effect of GNA may involve the regulation of the retinoid signaling pathway by butyrate in a retinoid-independent manner.

Objective: To investigate whether cardiac expression of the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is altered in patients with hypertensive heart disease (HHD)., Methods: We studied endomyocardial septal biopsies from 24 patients with essential hypertension divided into three groups: 6 without left ventricular hypertrophy (LVH) (HT group), 10 with LVH (LVH group), and 8 with LVH and heart failure (HF) (HF group). The expression of two PPARalpha isoforms (the native active and the truncated inhibitory) was analyzed by Western blot and reverse transcription polymerase chain reaction (RT-PCR), and two PPARalpha target genes were evaluated by RT-PCR. Histomorphological features were evaluated in a second myocardial sample from LVH and HF groups., Results: Whereas the expression of native PPARalpha protein was lower (p<0.05) in LVH and HF groups than in the HT group, truncated PPARalpha protein was overexpressed (p<0.001) in the HF group as compared with LVH and HT groups. The mRNA expression of native and truncated PPARalpha was similar in the three groups of hypertensives. In addition, a progressive decrease (p for trend<0.05) in the two PPARalpha target genes mRNA expression was observed among HT, LVH and HF groups. The amount of truncated PPARalpha protein correlates directly with cardiomyocytes apoptosis and inversely with cardiomyocytes density in patients with HHD. In addition, the expression of truncated PPARalpha protein was directly correlated with left ventricular volumes, and inversely with ejection fraction in all hypertensives., Conclusions: These findings suggest that post-transcriptional regulation of PPARalpha isoforms is altered in patients with HHD, namely in those developing HF. An excess of the truncated inhibitory isoform may be involved in hypertensive left ventricular failure and remodeling.

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.