Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor β (RXRβ)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRβ was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRβ was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRβ-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development.

Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis.

Use of all-trans retinoic acid (ATRA) as a differentiation agent has been limited to acute promyelocytic leukemia (APL) as non-APL leukemias are insensitive to ATRA. We recently demonstrated that the rexinoid, bexarotene, induces differentiation and therapeutic responses in patients with refractory AML. Rexinoids bind and activate retinoid X receptors (RXRs); however, rexinoids alone are incapable of activating retinoic acid receptor (RAR)/RXR complexes, suggesting that myeloid differentiation can occur independent of RAR. In this study, we demonstrate that rexinoid differentiation of AML cells is RAR independent and requires the expression of PU.1. Because of the promiscuousness of RXR with other nuclear receptors, myeloid differentiation by bexarotene with other nuclear receptor ligands was explored. Bexarotene cooperated with ATRA to enhance differentiation in some AML cell lines; however, the combination of bexarotene with the PPARγ agonist rosiglitazone did not. In contrast, bexarotene combined with liver X receptor (LXR) agonists, T0901317 or GW3965, induced potent differentiation and cytotoxicity in AML cell lines and primary human AML cells, but not in normal progenitor cells. These results suggest that RXR/LXR-regulated gene expression in normal cells is deregulated in AML cells and identifies a potential role for these agonists in differentiation therapy of non-APLs.

Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

Retinoic acid (RA) signaling is important for multiple aspects of embryonic development and tissue homeostasis. Heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) transduce RA signaling. It is not yet clear how the diversity of receptor combinations relates to the diversity of functions for RA. The expression patterns of three zebrafish RARs and four RXRs were reported recently. Here, we identify an additional RAR, a zebrafish RARgamma paralog, and two additional RXRs, duplicates of the previously identified RXRalpha and RXRgamma. Thus, the zebrafish genome contains duplicates of each RAR and RXR gene. All zebrafish RAR and RXR paralogs have overlapping and distinct areas of expression, as might be expected for duplicate genes in the process of diverging in function. By representing what is potentially the complete set of zebrafish RARs and RXRs, this study provides a valuable reference for future functional studies of the individual zebrafish RARs and RXRs.

2,4-Di-tert-butylphenol (2,4-DTBP) is an important commercial antioxidant and a toxic natural secondary metabolite that has been detected in humans. However, there is scant information regarding its toxicological effects. We asked whether 2,4-DTBP is a potential obesogen. Using a human mesenchymal stem cell adipogenesis assay, we found that exposure to 2,4-DTBP led to increased lipid accumulation and expression of adipogenic marker genes. Antagonist assays revealed that 2,4-DTBP increased lipid accumulation by activating the peroxisome proliferator-activated receptor (PPAR) γ-retinoid X receptor (RXR) heterodimer. 2,4-DTBP likely activated the PPARγ/RXRα heterodimer by activating RXRα but not directly binding to PPARγ. We confirmed that 2,4-DTBP directly bound to RXRα by solving the crystal structure of this complex, then predicted and demonstrated that related compounds could also activate RXRα. Our study demonstrated that 2,4-DTBP and related chemicals could act as obesogens and endocrine disruptors via RXRs. These data showed that 2,4-DTBP belongs to a family of compounds whose endocrine-disrupting and obesogenic effects can be strongly modulated by their chemical composition. Structure-activity studies such as the present one could help guide the rational development of safer antioxidants that do not interact with important nuclear receptors having broad effects on human development and physiology.

Transcriptome analyses performed in both human and zebrafish indicate strong expression of Apoe and Apoc1 by microglia. Apoe expression by microglia is well appreciated, but Apoc1 expression has not been well-examined. PPAR/RXR and LXR/RXR receptors appear to regulate expression of the apolipoprotein gene cluster in macrophages, but a similar role in microglia in vivo has not been studied. Here, we characterized microglial expression of apoc1 in the zebrafish central nervous system (CNS) in situ and demonstrate that in the CNS, apoc1 expression is unique to microglia. We then examined the effects of PPAR/RXR and LXR/RXR modulation on microglial expression of apoc1 and apoeb during early CNS development using a pharmacological approach. Changes in apoc1 and apoeb transcripts in response to pharmacological modulation were quantified by RT-qPCR in whole heads, and in individual microglia using hybridization chain reaction (HCR) in situ hybridization. We found that expression of apoc1 and apoeb by microglia were differentially regulated by LXR/RXR and PPAR/RXR modulating compounds, respectively, during development. Our results also suggest RXR receptors could be involved in endogenous induction of apoc1 expression by microglia. Collectively, our work supports the use of zebrafish to better understand regulation and function of these apolipoproteins in the CNS.

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a "non-cell autonomous" manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a "cell autonomous" manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma.

Vitamin A (VitA), via its active metabolite retinoic acid (RA), is critical for the maintenance of memory function with advancing age. Although its role in Alzheimer's disease (AD) is not well understood, data suggest that impaired brain VitA signaling is associated with the accumulation of β-amyloid peptides (Aβ), and could thus contribute to the onset of AD.

Peroxisome proliferator-activated receptors (PPARs) and their transcriptional partner retinoid X receptor (RXR) are involved in transcriptionally regulating the events that contribute to the control of parturition in humans. Definitive data, however, are lacking with respect to PPAR and RXR expression and activation during term labour in human gestational tissues. The aim of this study, therefore, was to identify tissue and labour-associated changes of PPAR isoforms (alpha, delta and gamma) and RXRalpha in placenta, amnion and choriodecidua. Gestational tissues from term non-labouring women were used for immunohistochemistry localisation and confirmation studies of PPAR isoforms (alpha, delta and gamma) and RXRalpha. Human gestational tissues were then collected from term women not-in-labour (NIL) (elective Caesarean section), in-labour (IL) (emergency Caesarean section) and post-labour (PL) (normal vaginal delivery). Quantitative RT-PCR (qRT-PCR) and Western blotting were employed to study mRNA and protein expression profiles respectively. Significantly higher mRNA expression was observed in placental tissues taken from women in labour (PPARdelta, PPARgamma and RXRalpha). Elevated PPARdelta and RXRalpha mRNA expression in fetal membranes was also associated with being in labour. In contrast, PPARgamma mRNA in the amnion was decreased with term PL compared to NIL. In placenta, PPARalpha, PPARdelta and PPARgamma protein expression was significantly increased in the IL group compared to the NIL or PL group. There was no significant difference in PPAR or RXRalpha protein expression in both amnion and choriodecidua between the three labour groups. PPAR (alpha and gamma) transcription factor DNA binding activity was found to decline IL compared to NIL and PL in the placenta. PPARdelta DNA binding activity also decreased in the choriodecidua IL compared to PL. In amnion, PPARalpha DNA binding activity was found to be higher IL compared to NIL. In conclusion, term human labour is associated with changes in expression and activity of PPAR isoforms and its transcription partner, RXRalpha. This data is consistent with the hypothesis that PPAR:RXR are involved in regulating of the processes of human term parturition.

Retinoid X receptors (RXR) are transcription factors with important roles in development, reproduction, homeostasis, and cell differentiation. Different types of vertebrate RXRs (α (RXRA), ß (RXRB) and γ (RXRG)) have arisen from multiple duplication events. The adaptive evolution mechanism that has preserved duplicate RXR paralogs, as well as their role in development and adaptation, is thus far unknown. In this work, we have investigated different aspects of vertebrate RXR evolution. Codon based tests of positive selection identified that RXR was under significant positive selection immediately after the whole genome duplications in vertebrates. Amino acid based rate shift analysis also revealed significant rate shifts immediately after the whole genome duplications and functional divergence between all the pairs of RXRs. However, the extant RXR genes are highly conserved, particularly the helix involved in dimerization and the DNA-binding domain, but positively selected sites can nevertheless be found in domains for RXR regulation.

Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3' positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements.

Retinoid X receptors (RXRs) are members of the nuclear receptor (NR) superfamily that mediate signalling by 9-cis retinoic acid, a vitamin A derivative. RXRs play key roles not only as homodimers but also as heterodimeric partners, e.g., for retinoic acid receptors, vitamin D receptors, and peroxisome proliferator-activated receptors. The NR family may also play important roles in the development of emphysema. However, the role of RXRs in the pathogenesis of emphysema is not well defined.

Aquatic organisms are exposed to diverse xenobiotics that cause peroxisome proliferation and/or endocrine disruption, both modulated in vertebrates by transcription factors of the nuclear receptor (NR) superfamily. Peroxisome proliferators are agonists of peroxisome proliferator-activated receptors (PPARs) that heterodimerize with the retinoid X receptor (RXR). Many xenoestrogens activate the estrogen receptor (ER). Here, 1090 bp of PPARalpha, 1255 bp of PPARgamma, 278 bp of RXRalpha, and 578 bp of ERalpha of thicklip grey mullet Chelon labrosus were cloned. Sequences were highly conserved, although relevant changes with respect to mammalian homologs were identified in PPARgamma and ERalpha. Semi-quantitative RT-PCR was used to determine if these NRs were expressed in different tissues of male, female and undifferentiated mullets captured in January and June. Expression of PPARs was highest in liver and lowest in muscle. RXRalpha expression was homogeneous excepting a low expression in male and female gill in January and brain and heart of undifferentiated fish in January and June. ERalpha expression predominated in liver and female gonad in June. The expression level of PPARs and ERalpha was significantly higher in liver in January than in gills in January or June. The present results show tissue-dependent modulation of expression of NRs in mullets.

Ginseng has a wide range of beneficial effects on health, such as the mitigation of minor and major inflammatory diseases, cancer, and cardiovascular diseases. There are abundant data regarding the health-enhancing properties of whole ginseng extracts and single ginsenosides; however, no study to date has determined the receptors that mediate the effects of ginseng extracts. In this study, for the first time, we explored whether the antiinflammatory effects of Rg3-enriched red ginseng extract (Rg3-RGE) are mediated by retinoid X receptor α-peroxisome-proliferating receptor γ (RXRα-PPARγ) heterodimer nuclear receptors.

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3-5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5-4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5' UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.

The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR). We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo. Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene. Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha. Homodimeric interaction by mPPAR was not detected. These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.

Nuclear receptors (NRs) are transcription factors accomplishing a multiplicity of functions, essential for organismal homeostasis. Among their numerous members, the retinoid X receptor (RXR) is a central player of the endocrine system, with a singular ability to operate as a homodimer or a heterodimer with other NRs. Additionally, RXR has been found to be a critical actor in various processes of endocrine disruption resulting from the exposure to a known class of xenobiotics termed organotins (e.g., tributyltin (TBT)), including imposex in gastropod molluscs and lipid perturbation across different metazoan lineages. Thus, given its prominent physiological and endocrine role, RXR is present in the genomes of most extant metazoan species examined to date. Here, we expand on the phylogenetic distribution of RXR across the metazoan tree of life by exploring multiple next-generation sequencing projects of protostome lineages. By addressing amino acid residue conservation in combination with cell-based functional assays, we show that RXR induction by 9-cis retinoic acid (9cisRA) and TBT is conserved in more phyla than previously described. Yet, our results highlight distinct activation efficacies and alternative modes of RXR exploitation by the organotin TBT, emphasizing the need for broader species sampling to clarify the mechanistic activation of RXR.

Abnormal signaling by retinoids or n-3 polyunsaturated fatty acids has been implicated in clinical depression. The converging point in activities of these two classes of molecules is transcriptional activation of retinoid X receptors (Rxr). We show here that ablation of Rxrgamma in mice leads to depressive-like behaviors including increased despair and anhedonia, which were accompanied by reduced expression of dopamine D2 receptor in the shell of nucleus accumbens (NAc) and altered serotonin signaling. While abnormal serotonin signaling is not sufficient to generate the depressive behaviors, increasing D2r expression by chronic fluoxetine (Prozac) treatment or adenoassociated virus type2 (AAV2) mediated expression of Rxrgamma or D2r in the NAc of Rxrgamma(-/-) mice normalizes depressive-like behaviors in Rxrgamma(-/-) animals. Conversely, NAc infusion of raclopride, a D2r antagonist prevents AAV2-Rxrgamma-mediated rescue of despair behaviors in Rxrgamma(-/-) mice. Combined, our data argue that control of NAc D2r expression is critical for Rxrgamma-mediated modulation of affective behaviors.

The experimental activation of retinoid receptors reduces pathological symptoms in animal models of multiple sclerosis. In order to assess the involvement of endogenous retinoid signaling during the process of demyelination we investigated retinoic acid synthesizing enzymes and nuclear receptors using the mouse model of cuprizone toxicity. The initiation of myelin degradation in the corpus callosum was accompanied with a local increase of retinaldehyde dehydrogenase (RALDH) immunoreactivity. On the level of receptors we observed a striking increase in protein expression of the retinoid X receptor (RXR)-β in the affected corpus callosum. The RXRβ immunoreactivity appeared exclusively in astrocytes, where it reached a maximum at five weeks of treatment, following the RALDH response. In the cerebral cortex and basal ganglia of affected mice RXRβ was also observed in neurons. Among nuclear receptor antigens RARα showed a cuprizone associated increase in the corpus callosum. Quantitative RT-PCR revealed strong basal expression of RXRβ and a significant, over 20-fold upregulation of the peroxisome proliferator-activated receptor-γ during demyelination. The results indicate that compensatory mechanisms during central demyelination may engage nuclear receptor dimers with an RXRβ partner.