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Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility.

Background: Glycogen synthase kinase-3β (GSK-3β) is recently demonstrated to be a tumor promoter and a potential drug target in several tumors. However, the implication and mechanism of GSK-3β in hepatocellular carcinoma (HCC) remain unexplored. Methods: We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we investigated where GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Coimmunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to study the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments. Findings: We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition: from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ. Interpretation: Our findings suggest that overexpression of GSK-3β may confer HCC growth through interfering retinoid signalling. Targeting GSK3β can be a promising strategy for designing improved treatment of sorafenib in HCC. Funding Statement: This work is supported by grants from Natural Science Foundation of China (No. 81673467, 31471273, 31461163002/RGC N_HKU 740/14), the 10th Singapore-China Joint Research Program (S2014GR0448), Natural Science Foundation of Fujian Province (2019I0002) and Leading Talents in Scientific and Technological Innovation, Double Hundred Talents Program of Fujian Province. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All the use of human samples and study protocols were approved by the Hospital Ethics Committee. All patients signed an informed consent form in prior to sample collection.

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.

MicroRNAs (miRNAs) play critical roles in the complex networks of virus–host interactions. Our previous research showed that porcine reproductive and respiratory syndrome virus (PRRSV) infection markedly upregulates miR-c89 expression, suggesting that miR-c89 may play an important role in PRRSV infection. The present study sought to determine the function of miR-c89 and its molecular mechanism during PRRSV infection. Using quantitative reverse transcription PCR (RT-qPCR) verification, we demonstrated that both highly pathogenic PRRSV and low-pathogenic PRRSV infection induced miR-c89 expression. The overexpression of miR-c89 significantly suppressed the replication of a variety of PRRSV strains, regardless of the timing of infection. Further, miR-c89 can directly target the 3′UTR of porcine retinoid X receptor β (RXRB) mRNA in a sequence-specific manner. Knockdown affected RXRB expression, as siRNA can suppress the replication of a variety of PRRSV strains. This work not only provides new insights into PRRSV–cell interactions, but also highlights the potential for the use of miR-c89 in the development of new antiviral strategies to combat PRRSV infection.

Macrophages play important and diverse roles during cancer progression. However, cancer therapies based on macrophage modulation are lacking in tools that can recognize and deliver therapeutic payloads to macrophages in a tumor-specific manner. As a result, treatments tend to interfere with normal macrophage functions in healthy organs. We previously identified a macrophage-binding peptide, termed CRV. Here, we show that upon systemic administration into tumor-bearing mice, CRV selectively homes to tumors, extravasates, and preferentially binds to macrophages within. CRV exhibits a higher affinity for tumor macrophages than for other cells in tumors or for other macrophage types elsewhere in the body. We further identified and validated retinoid X receptor beta (RXRB) as the CRV receptor. Intriguingly, although it is known as a nuclear receptor, RXRB shows a prominent cell surface localization that is largely restricted to tumor macrophages. Systemic administration of anti-RXRB antibodies also results in tumor-selective binding to macrophages similar to CRV. Lastly, we demonstrate the ability of CRV to improve the delivery of nano-carriers into solid tumors and macrophages within. In summary, we describe here a novel cell surface marker and targeting tools for tumor macrophages that may aid in future development of macrophage-modulatory cancer therapies.

Hepatic stellate cells are liver-specific perivascular cells, identified as the major source of collagen in liver fibrosis, following their activation and conversion to myofibroblast-like cells. Lycopene is a carotenoid with biological activities and protective effects described in different pathologies, but little is known about its role in liver protection. We evaluated the influence of lycopene on the cell cycle and lipid metabolism and monitored the possible pathways involved in lycopene inhibition of stellate cell activation. Lycopene induced expression of the lipocyte phenotype, with an accumulation of fat droplets in cytoplasm, with high synthesis and turnover of phospholipids and triglycerides. Cell proliferation analysis showed that lycopene reduced the growth of GRX cells. Lycopene induced an arrest in the G0/G1 phase, followed by a decrease of cells in the G2/M phase, regardless of the concentration of lycopene used. Lycopene modulated relevant signaling pathways related to cholesterol metabolism, cellular proliferation, and lipid metabolism. Also, lycopene treatment increased the expression of RXR-α, RXR-β, and PPARγ, important biomarkers of liver regeneration. These results show that lycopene was able to negatively modulate events related to the activation of hepatic stellate cells through mechanisms that involve changes in expression of cellular lipid metabolism factors, and suggest that this compound might provide a novel pharmacological approach for the prevention and treatment of fibrotic liver diseases.

Retinoid X receptor beta (RXRβ) has been poorly studied in atherosclerosis. The aim of the present study is to explore the function of RXRβ in oxidized low density lipoprotein (ox-LDL)-induced inflammation in endothelial cells and the underlying mechanism. The protein expression of RXRβ in the aorta of atherosclerotic mice was detected. A lentivirus vector for RXRβ overexpression and RNA interference for RXRβ downregulation were constructed and transfected into human aortic endothelial cells (HAECs). The results showed that RXRβ protein expression was downregulated in aorta of high fat diet (HFD)-fed LDLr-/- mice and ox-LDL-treated HAECs. The ox-LDL-induced production of pro-inflammatory cytokines and activations of TLR9/NF-κB and NLRP3/caspase-1 inflammasome pathway were significantly decreased by RXRβ overexpression but increased by RXRβ knockdown in HAECs. The ox‑LDL‑induced mitochondrial damage indicated as the increased generation of mitochondrial ROS, decreased mitochondrial membrane potential and increased mitochondrial DNA release was abolished by RXRβ overexpression but aggravated by RXRβ knockdown. Treatment with mito-TEMPO significantly reduced the increased production of pro-inflammatory cytokines and activations of TLR9/NF-κB and NLRP3/caspase-1 inflammasome induced by RXRβ knockdown in ox-LDL treated HAECs. Moreover, peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) protein expression was reduced in HFD-fed LDLr-/- mice. RXRβ could interact with PGC1α in HAECs. Ox-LDL-induced reduction of PGC1α was significantly inhibited by RXRβ overexpression and aggravated by RXRβ downregulation. Our further study showed that transfection of PGC1α siRNA abrogated the alleviative effects of RXRβ overexpression on mitochondrial damage and inflammation in ox-LDL treated cells. The present study indicates that RXRβ exerted protective effects against the ox-LDL-induced inflammation may through regulating PGC1α-dependent mitochondrial homeostasis.

Background The tumor immune microenvironment is pivotal in predicting clinical outcomes and therapeutic efficacy in cancer patients. This study aims to develop an immune prediction model (IPM) to effectively predict prognosis and immunotherapeutic response in patients with hepatocellular carcinoma (HCC). Methods An IPM was constructed and validated based on immune-related genes. The influence of IPM on the HCC immune microenvironment, as well as the possible mechanism, was comprehensively analyzed. The value of the model in predicting the response of HCC patients to immunotherapy was also evaluated. Results A novel IPM based on eight genes was developed and validated to predict the prognosis of HCC patients. These genes are matrix metalloproteinase 12 (MMP12), heme oxygenase 1 (HMOX1), C-X-C motif chemokine receptor 6 (CXCR6), hepatoma-derived growth factor (HDGF), placental growth factor (PGF), tyrosine kinase 2 (TYK2), retinoid X receptor beta (RXRB), and cyclin-dependent kinase 4 (CDK4). High-risk patients showed significantly poorer survival than low-risk patients. A nomogram was also established based on the IPM and tumor, node, metastasis (TNM) classification, which showed some net clinical benefit. Gene set enrichment analysis (GSEA) revealed several significantly enriched oncological signatures and immunologic signatures. Furthermore, high-risk patients were characterized by severe clinicopathological characteristics and immune cell infiltration. Finally, we found the that the IPM showed a significant positive correlation with programmed cell death 1 (PDCD1), cluster of differentiation 274 (CD274), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression, suggesting a potentially enhanced effects of immunotherapy antibodies in HCC patients with a high risk score. Conclusions A novel IPM that could predict clinical prognosis and immunotherapeutic response in HCC patients was developed. Our findings not only provide new insights into the identification of HCC patients with poor survival, but also deepen our understanding of the immune microenvironment, as well as the mechanism of immunotherapy, in HCC.

Multiple myeloma (MM) is an incurable hematologic malignancy due to inevitable relapse and chemoresistance development. Our preliminary data show that MM cells express high levels of PGC1β and LDHA. In this study, we investigated the mechanism behind PGC1β‐mediated LDHA expression and its contribution to tumorigenesis, to aid in the development of novel therapeutic approaches for MM. Real‐time PCR and western blotting were first used to evaluate gene expression of PGC1β and LDHA in different MM cells, and then, luciferase reporter assay, chromatin immunoprecipitation, LDHA deletion report vectors, and siRNA techniques were used to investigate the mechanism underlying PGC1β‐induced LDHA expression. Furthermore, knockdown cell lines and lines stably overexpressing PGC1β or LDHA lentivirus were established to evaluate in vitro glycolysis metabolism, mitochondrial function, reactive oxygen species (ROS) formation, and cell proliferation. In addition, in vivo xenograft tumor development studies were performed to investigate the effect of PGC1β or LDHA expression on tumor growth and mouse survival. We found that PGC1β and LDHA are highly expressed in different MM cells and LDHA is upregulated by PGC1β through the PGC1β/RXRβ axis acting on the LDHA promoter. Overexpression of PGC1β or LDHA significantly potentiated glycolysis metabolism with increased cell proliferation and tumor growth. On the other hand, knockdown of PGC1β or LDHA largely suppressed glycolysis metabolism with increased ROS formation and apoptosis rate, in addition to suppressing tumor growth and enhancing mouse survival. This is the first time the mechanism underlying PGC1β‐mediated LDHA expression in multiple myeloma has been identified. We conclude that PGC1β regulates multiple myeloma tumor growth through LDHA‐mediated glycolytic metabolism. Targeting the PGC1β/LDHA pathway may be a novel therapeutic strategy for multiple myeloma treatment.

Per- and polyfluoroalkyl substances (PFAS) are a broad class of hundreds of fluorinated chemicals with environmental health concerns due to their widespread presence and persistence in the environment. Several of these chemicals have been comprehensively studied for experimental toxicity, environmental fate and exposure, and human epidemiology; however, most chemicals have limited or no data available. To inform methods for prioritizing these data-poor chemicals for detailed toxicity studies, we evaluated 142 PFAS using an in vitro screening platform consisting of two multiplexed transactivation assays encompassing 81 diverse transcription factor activities and tested in concentration-response format ranging from 137 nM to 300 μM. Results showed activity for various nuclear receptors, including three known PFAS targets--specifically estrogen receptor alpha and peroxisome proliferator receptors alpha and gamma. We also report activity against the retinoid X receptor beta, the key heterodimeric partner of type II, non-steroidal nuclear receptors. Additional activities were found against the pregnane X receptor, nuclear receptor related-1 protein, and nuclear factor erythroid 2-related factor 2, a sensor of oxidative stress. Using orthogonal assay approaches, we confirmed activity of representative PFAS against several of these targets. Finally, we identified key PFAS structural features associated with nuclear receptor activity that can inform future predictive models for use in prioritizing chemicals for risk assessment and in the design of new structures devoid of biological activity.

Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55 kDa), RXRβ (47 kDa) and RXR (56 kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis.

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.

Since the isolation and identification of the retinoid X receptor (RXR) as a member of the nuclear receptor (NR) superfamily in 1990, its analysis has ushered in a new understanding of physiological regulation by nuclear receptors, and novel methods to identify other unknown and orphan receptors. Expression of one or more of the three isoforms of RXR-α, β, and γ-can be found in every human cell type. Biologically, RXR plays a critical role through its ability to partner with other nuclear receptors. RXR is able to regulate nutrient metabolism by forming "permissive" heterodimers with peroxisome proliferator-activated receptor (PPAR), liver-X-receptor (LXR), farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which function when ligands are bound to one or both of the heterodimer partners. Conversely, RXR is able to form "nonpermissive" heterodimers with vitamin D receptor (VDR), thyroid receptor (TR) and retinoic acid receptor (RAR), which function only in the presence of vitamin D, T3 and retinoic acid, respectively. Furthermore, RXR can form homodimers in the presence of a selective agonist, or rexinoid, to regulate gene expression and to either inhibit proliferation or induce apoptosis in human cancers. Thus, over the last 25 years there have been several reports on the design and synthesis of small molecule rexinoids. This review summarizes the synthetic methods for several of the most potent rexinoids thus far reported.

Summary 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.

Background/Aims: Vitamin A (VA) protects the intestinal epithelial barrier by improving cell migration and proliferation. Our previous studies demonstrated that VA deficiency (VAD) during pregnancy suppresses the systemic and mucosal immune responses in the intestines of offspring and that VA supplementation (VAS) during early life can increase immune cell counts. However, little is known about the mechanisms by which VA regulates intestinal epithelial barrier function. Methods: Caco-2 cells were treated with all-trans retinoic acid (ATRA) for 24 hours to determine the optimum ATRA concentration to which the cells in question respond. Caco-2 cells were infected with recombinant adenoviruses carrying retinoic acid receptor beta (Ad-RARβ) and small interfering RARβ(siRARβ) to assess the effects of RARβ signalling on the expression of specific proteins. A siTLR4 lentivirus was used to knockdown Toll-like receptor 4 (TLR4) in Caco-2 cells to determine its role in the protective effects of VA on the intestinal epithelial barrier, and experiments involving TLR4-knock-out mice were performed to verify the effect of TLR4. VA normal (VAN), VAD and VAS rat models were established to confirm that changes in RARβ, TLR4 and ZO-2 expression levels that occurred following decreases or increases in retinol concentrations in vivo, and the permeability of the Caco-2 cell monolayer, as well as that of the epithelial barrier of the rat intestine was detected by measuring transepithelial resistance (TER) or performing enzyme-linked immunosorbent assay (ELISA). Retinoic acid receptor (RAR), toll like receptor (TLR) and tight junction (TJ) mRNA and protein expression levels in Caco-2 cells and the colon monolayers in the rat and mouse models were measured by PCR and western blotting, respectively. Co-immunoprecipitation (co-IP) and immunofluorescence staining were performed to assess the interactions among RARβ, TLR4 and zonula occluden-2 (ZO-2) in Caco-2 cells, and chromatin immunoprecipitation (ChIP) assay was performed to assess the binding between RARβ and the TLR4 promoter sequence in Caco-2 cells. Results: In the present study, ATRA treatment not only increased the TER of the Caco-2 monolayer but also up-regulated the expression levels of RARβ, TLR4 and ZO-2 in Caco-2 cells. The expression levels of these three proteins were significantly decreased in the colonic epithelial monolayers of VAD rats compared with those of VAN rats and were significantly increased following VAS in the corresponding group compared with the control group. Furthermore, the above changes in TLR4 and ZO-2 expression levels were augmented or attenuated by Ad-RARβ or siRARβ infection, respectively, in Caco-2 cells. Interestingly, siTLR4 down-regulated ZO-2 expression but did not affect RARβ expression in Caco-2 cells, and in VAD mice the lack of TLR4 did not affect ZO-2 expression. We noted direct interactions between RARβ and TLR4, TLR4 and ZO-2 in Caco-2 cells, and ChIP assay showed that RARβ could bind to the TLR4 promoter but not the ZO-2 promoter in Caco-2 cells. Conclusion: Taken together, our results indicate that RARβ enhanced ZO-2 expression by regulating TLR4 to improve intestinal epithelial barrier function in Caco-2 cells, as well as in rat and mouse models, but not in humans.

Stroke is the consequence of limited blood flow to the brain with no established treatment to reduce the neurological deficits. Focusing on therapeutic protocols in targeting subventricular zone (SVZ) neurogenesis has been investigated recently. This study was designed to evaluate the effects of retinoic acid (RA)-pretreated Wharton's jelly mesenchymal stem cells (WJ-MSCs) in combination with triiodothyronine (T3) in the ischemia stroke model. Male Wistar rats were used to induce focal cerebral ischemia by middle cerebral artery occlusion (MCAO). There were seven groups of six animals: Sham, Ischemic, WJ-MSCs, RA-pretreated WJ-MSCs, T3, WJ-MSCs +T3, and RA-pretreated WJ-MSCs + T3. The treatment was performed at 24 h after ischemia, and animals were sacrificed one week later for assessments of retinoid X receptor β (RXRβ), brain-derived neurotrophic factor (BDNF), Sox2 and nestin in the SVZ. Pro-inflammatory cytokines in sera were measured at days four and seven after ischemia. RXRβ, BDNF, Sox2 and nestin had the significant expressions in gene and protein levels in the treatment groups, compared with the ischemic group, which were more vivid in the RA-pretreated WJ-MSCs + T3 (p ≤ 0.05). The same trend was also resulted for the levels of TNF-α and IL-6 at four days after ischemia (p ≤ 0.05). In conclusion, application of RA-pretreated WJ-MSCs + T3 could be beneficial in exerting better neurotrophic function probably via modulation of pro-inflammatory cytokines.

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.

Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.

Purpose The purpose of this study was to investigate the influence of selective agonists of the retinoid receptor X (RXR) and the retinoid acid receptor (RAR) on bone metabolism in rats. Methods Thirty six male Wistar rats were divided into three groups: receiving bexarotene, or tazarotene, or to control group. Serum biochemical markers of bone turnover (osteocalcin – OC, tartrate resistant acid phosphatase 5 – TRACP5b and osteoprotegerin – OPG) and mechanical properties of bones were analyzed. Results There was a significant decrease in the femur index value in groups receiving tazarotene and bexarotene on Day 14 (8% and 20% respectively, p = 0.0039). On Day 28, 14 days after discontinuation of tazarotene and bexarotene, the difference in femur indexes was still significant (4% for T1–6 and B1–6, p = 0.0270). In the bexarotene group an increase in mean plasma osteocalcin level and mean plasma TRACP5b level was detected. In the tazarotene group the mean osteocalcin level remained unchanged and the mean plasma TRACP5b level decreased. An increased yield stress was detected in groups receiving retinoids comparing to controls after 14 days of tazarotene and bexarotene administration. Conclusion Although bexarotene and tazarotene administration caused decrease in the femur index, mechanisms responsible for that effect seem to be different. Our results suggest that bexaroten increases bone turnover. On the contrary, tazaroten seems to have inhibitory effect on bone turnover. A counter influence of selective RAR and RXR agonists on the bone turnover might be the reason for inconsistency in results from published research concerning the influence of retinoids on bone metabolism.

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus.

Retinoic acid (RA), an active metabolite of vitamin A (VA), is important for many physiological processes including energy metabolism. This is mainly achieved through RA-regulated gene expression in metabolically active cells. RA regulates gene expression mainly through the activation of two subfamilies in the nuclear receptor superfamily, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RAR/RXR heterodimers or RXR/RXR homodimers bind to RA response element in the promoters of RA target genes and regulate their expressions upon ligand binding. The development of metabolic diseases such as obesity and type 2 diabetes is often associated with profound changes in the expressions of genes involved in glucose and lipid metabolism in metabolically active cells. RA regulates some of these gene expressions. Recently, in vivo and in vitro studies have demonstrated that status and metabolism of VA regulate macronutrient metabolism. Some studies have shown that, in addition to RARs and RXRs, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter-transcription factor II, and peroxisome proliferator activated receptor β/δ may function as transcriptional factors mediating RA response. Herein, we summarize current progresses regarding the VA metabolism and the role of nuclear receptors in mediating RA signals, with an emphasis on their implication in energy metabolism.

We review trialkyltin and triaryltin compounds, representing a class of organometallic compounds that function as nuclear retinoid X receptors (RXR) agonists due to their capability to bind to the ligand-binding domain of RXR subtypes and function as transcriptional activators. RXRs act predominantly as heterodimers with other nuclear receptors as permissive heterodimers with peroxisome proliferator-activated receptors, liver X receptors, farnesoid X receptor, pregnane X receptor and constitutive androstan receptor or as non-permissive heterodimer with vitamin D receptor, and as conditional heterodimers with retinoid receptors, and thyroid hormone receptors. RXR – “partner” receptor heterodimers are considered to be ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. Tributyltin at even pico- or nanomolar concentrations may cause the superimposition of male genitalia on female in several aquatic organisms, since they are DNA-targeted, mitotic, and their actions are occurring through target gene(s)-mediated pathways. They may cause molecular interactions with reproductive system in mammals, and as potent environmental obesogens, they promote adipocyte differentiation. Organotin compounds become known also for their immunotoxicity, neurotoxicity, for their effects on reproduction and/or development. We also review effects of organotins with respect to levels and activities of hepatic P450s and aromatase activity.

Fourteen novel compounds were prepared and their antagonistic activities against liver X receptors (LXR) α/β were tested in vitro. Compound 26 had an IC50 value of 6.4 µM against LXRα and an IC50 value of 5.6 µM against LXRβ. Docking studies and the results of structure-activity relationships support the further development of this chemical series as LXRα/β antagonists.

All-trans retinoic acid (ATRA) triggers a wide range of effects on vertebrate development by regulating cell proliferation, differentiation, and apoptosis. ATRA activates retinoic acid receptors (RARs) which heterodimerize with retinoid X receptors (RXRs). RAR/RXR heterodimers function as ATRA-dependent transcriptional regulators by binding to retinoic acid response elements (RAREs). To identify RAR/RXR heterodimer-binding sites in the human genome, we performed a modified yeast one-hybrid assays and identified 193 RAR/RXR heterodimer-binding fragments in the human genome. The putative target genes included genes involved in development process and cell differentiation. Gel mobility shift assays indicated that 160 putative RAREs could directly interact with the RAR/RXR heterodimer. Moreover, 19 functional regulatory single nucleotide polymorphisms (rSNPs) on the RAR/RXR-binding sequences were identified by analyzing the difference in the DNA-binding affinities. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of RAR/RXR heterodimers.

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

Retinoic acid functions through two classes of receptors, i.e., the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). The difference in the role between RAR and RXR, however, are not well clarified. In the present study, we comparatively investigated the phenotypic and global gene expression changes in Xenopus tropicalis embryos induced by three different agonists, including a RAR selective ligand (all- trans retinoic acid, at-RA), a RXR selective ligand (fluorobexarotene, FBA) and their common ligand (9- cis retinoic acid, 9c-RA). All three agonists induced striking and similar malformations in X. tropicalis embryos at the concentrations of 5–50 μg/L. Especially, the development of anterior structures and caudal region was dramatically altered. The hierarchical clustering analysis of phenotypes and gene profiles suggested that effects induced by 9c-RA separated from those by at-RA and FBA. The up-regulated genes were involved in multiple pathways while down-regulated genes were mainly related to phototransduction and tyrosine metabolism. at-RA primarily affected the retinol, glycolysis, starch and sucrose metabolisms while FBA led to disturbances in more wide-ranging pathways such as the PPAR, adipocytokine, insulin, FoxO signaling pathways, alanine, aspartate and glutamate metabolism. RXR is a heterodimeric partner for several other nuclear receptors, which opens the possibility that additional retinoid effects could be mediated by FBA, such as RXR-PPAR. Our data indicates that not only RXR-RAR but also RXR-PPAR plays important roles in the control of metabolism with retinoid treatment in X. tropicalis embryos.

The androgen receptor (AR) can be stimulated by interleukin-6 (IL-6) in the absence of androgens to induce prostate cancer progression. The purpose of this study was to investigate whether the co-activator steroid receptor coactivator-1 (SRC-1) and co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) are involved in IL-6-induced AR activation. The effects of IL-6 on LNCaP cell proliferation were monitored using real-time cell analysis (RTCA) iCELLigence system. The impacts of IL-6 on the association of the AR with SRC-1 and SMRT were investigated using the mammalian two-hybrid assay. IL-6 increased the proliferation of LNCaP cells with maximal induction at 50 ng/mL. The AR-SRC-1interaction was enhanced by IL-6, with maximal induction at the concentration of 50 ng/mL (P

Systemic sclerosis is a systemic autoimmune and connective tissue disorder associated with the human leukocyte antigen locus. However, the functional relationship between human leukocyte antigen gene(s) and disease development remains unknown. To elucidate major histocompatibility complex-linked systemic sclerosis genetics, we performed genotyping of major histocompatibility complex-borne microsatellites and HLA-DPB1 alleles using DNA obtained from 318 anti-topoisomerase I antibody-positive patients and 561 healthy controls, all of Japanese descent. Those results revealed two major histocompatibility complex haplotypes associated with systemic sclerosis. Exome sequencing and targeted analysis of these risk haplotypes identified rs17847931 in RXRB as a susceptibility variant ( P = 1.3 × 10 −15 ; odds ratio [OR] = 9.4) with amino acid substitution p.V95A on the risk haplotype harboring HLA-DPB1∗13:01 . No identical variant in the other haplotype including DPB1*09:01 was observed, though that haplotype also showed a significant association ( P = 8.5 × 10 −22 ; OR = 4.3) with systemic sclerosis. Furthermore, the number of risk factors was shown to be a predominant factor, as individuals with two factors had elevated risk ( P = 6.7 × 10 −13 ; OR = 30.2). We concluded that RXRB may be involved in antifibrotic activity in skin and chromatin remodeling.

Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)–mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, precluding its nuclear translocation. In addition, ERK, activated by AEG-1, phosphorylates RXR that leads to its functional inactivation and attenuation of ligand-dependent transactivation. In nude mice models, combination of all-trans retinoic acid (ATRA) and AEG-1 knockdown synergistically inhibited growth of human HCC xenografts. The present study establishes AEG-1 as a novel homeostatic regulator of RXR and RXR/RAR that might contribute to hepatocarcinogenesis. Targeting AEG-1 could sensitize patients with HCC and AML to retinoid- and rexinoid-based therapeutics. Cancer Res; 74(16); 4364–77. ©2014 AACR.

We exposed Xenopus tropicalis embryos to a selective antagonist of retinoid X receptor (UVI3003). UVI3003 induced multiple malformations at the concentrations of 200–1000 μg/L after 48 h exposure. The most prominent malformations affected brains, eyes, cement gland and fins. UVI3003 also induced variable and divergent malformations at 250–1500 μg/L after 0–24 and 24–48 h exposure. Microarray analysis showed that seven genes (rps15, serp2, fmr1, cyp2e1, lrrc9, ugtla6 and LOC100490188) were differentially regulated in all three treatment groups after 0–24 h exposure. The most significantly affected pathway was galactose metabolism. In 24–48 h exposure groups, 18 genes were differentially regulated, mainly comprising components of the PPAR signaling pathway. These results suggested that UVI3003 is teratogenic in amphibian embryos. Differential gene expression suggests that galactose metabolism and PPAR signaling pathways may provide underlying mechanistic detail accounting for the observed malformations.

Selective ligands for retinoic acid receptors (RARs) and for retinoid X receptors (RXRs) are required for both biological studies and therapeutic purposes. We have synthesized a series of diarylamines incorporating hexahydrophenalene or octahydrobenzoheptalene as a hydrophobic moiety and examined their activities towards RARs and RXRs. Most of these compounds showed agonistic activity towards RXRs, but were inactive towards RARs. These RXR-specific ligands showed synergistic activity in RARα,β ligand-induced terminal differentiation of leukemia cell line HL-60.

Scope β-Carotene (BC), a substitute for vitamin A, is widely used for its benefits. The present study investigates whether in-utero BC administration can alter lipid and glucose homoeostasis in offspring. Methods and results Pregnant mice are supplemented with BC (1 mg kg-1 weight) by oral gavage once every 3 days, for a total of six doses. Increased visceral fat may be caused by up-regulated PPARγ (peroxisome proliferator-activated receptor gamma) and RXRα/β (retinoid X receptors) in liver and adipose tissue, and glucose intolerance is observed in F1 adult females prenatally supplemented with BC, while F1 males do not exhibit these symptoms. In females, increased serum leptin, resistin, and IL-6 and reduced adiponectin, caused by visceral obesity, may result in downregulated insulin receptor signaling in muscle and further account for glucose intolerance. Increased pancreatic β-cell mass might compensate for the downregulated insulin gene (ins2). Increased glucagon and α-cell mass, accompanied by upregulated glucagon gene (gcg), might also be risk factors for the development of diabetes. Conclusions Maternal supplementation with BC disturbs lipid metabolism and induces glucose intolerance in F1 female mice, suggesting that BC supplementation during pregnancy should be used with caution.

How Retinoid X receptors (RXR) and thyroid hormone receptors (TR) interact on negative TREs and whether RXR subtype specificity is determinant in such regulations is unknown. In a set of functional studies, we analyzed RXR subtype effects in T3-dependent repression of hypothalamic thyrotropin-releasing hormone (Trh). Two-hybrid screening of a hypothalamic paraventricular nucleus cDNA bank revealed specific, T3-dependent interaction of TRs with RXRβ. In vivo chromatin immuno-precipitation showed recruitment of RXRs to the TRE-site 4 region of the Trh promoter in the absence of T3. In vivo overexpression of RXRα in the mouse hypothalamus heightened T3-independent Trh transcription, whereas RXRβ overexpression abrogated this activity. Loss of function of RXRα and β by shRNAs induced inverse regulations. Thus, RXRα and RXRβ display specific roles in modulating T3-dependent regulation of Trh. These results provide insight into the actions of these different TR heterodimerization partners within the context of a negatively regulated gene.

Three unreported analogues of 4-[1-(3,5,5,8,8-pentamethyl-5-6-7-8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), otherwise known as bexarotene, as well as four novel analogues of (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl)-4-hydroxyphenyl)acrylic acid (CD3254), are described and evaluated for their retinoid X receptor (RXR) selective agonism. Compound 1 has FDA approval as a treatment for cutaneous T-cell lymphoma (CTCL), although treatment with 1 can elicit side-effects by disrupting other RXR-heterodimer receptor pathways. Of the seven modeled novel compounds, all analogues stimulate RXR-regulated transcription in mammalian 2 hybrid and RXRE-mediated assays, possess comparable or elevated biological activity based on EC50 profiles, and retain similar or improved apoptotic activity in CTCL assays compared to 1. All novel compounds demonstrate selectivity for RXR and minimal crossover onto the retinoic acid receptor (RAR) compared to all-trans-retinoic acid, with select analogues also reducing inhibition of other RXR-dependent pathways (e.g., VDR-RXR). Our results demonstrate that further improvements in biological potency and selectivity of bexarotene can be achieved through rational drug design.

Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription. CTBP2 suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.

This exhaustive in silico study looks into the molecular interactions of phthalates and their metabolites with human peroxisome proliferator-activated receptor (hPPAR) and retinoid X receptor (hRXR) α, β and γ subtypes--the nuclear receptor proteins function as transcription factors by regulating the expression of downstream genes. Apart from the much discussed plasticizer bisphenol A, we examined the binding affinities of 15 common diphthalates and their monophthalates, natural (linoleic acid, conjugated linoleic acid) and synthetic (bezafibrate, pioglitazone, GW 50156) ligands with hPPARs. In addition to these phthalates, specific natural (retinoic and phytanic acids) and synthetic (bexarotene, rosiglitazone) ligands were examined with hRXRs. The Maestro, Schrodinger Suite 2012 was used for the molecular docking study. In general, natural ligands of hPPAR showed less binding efficiencies than phthalic acid esters and drugs. The diphthalate di-iso-decyl phthalate showed the highest G score (-9.99) with hPPAR (γ), while its monophthalate (mono-iso-decyl phthalate) showed a comparatively less G score (-9.56). Though the PPAR modulator GW 50156 showed strong affinity with all hPPAR subtypes, its highest G score (-12.43) was with hPPARβ. Hazardous di(2-ethylhexyl)phthalate generally showed a greater preference to hRXRs than hPPARs, but its highest G score (-10.87) was with hRXRα; while its monophthalate (Mono(2-ethylhexyl)phthalate) showed a lesser G score (-8.59). The drug bexarotene showed the highest G score (-13.32) with hRXRβ. Moreover, bisphenol A showed more affinity towards hRXR. Briefly, this study gives an overview on the preference of phthalic acid esters, natural and synthetic ligands on to hPPAR and hRXR subtypes, which would lead to further in vitro mechanistic as well as in vivo preclinical and clinical studies.

There is increasing evidence suggesting that fatty acids biosynthesis and metabolism are regulated by peroxisome proliferator-activated receptors (PPARs), mostly through the PPAR signaling pathway at the transcriptomic level. We hypothesized that the genetic variants of the enzymes in the PPAR signaling pathway may be associated with the traits of porcine meat quality (PMQ). We mined 77 potentially functional single nucleotide polymorphisms in the PPAR signaling pathway of the pig. There were 13 TagSNPs in 13 different genes mapped within the reported pig quantitative trait loci (QTLs) regions related to PMQ based on the Pig QTL database. Based on the association study with ten measured PMQ traits in both the pathway level and the SNP level, we tested eight significantly associated traits with additive effect in the PPAR signaling pathway and explored only one significant TagSNP in gene RXRB, which is directly associated with the trait of skin weight. Moreover, several interactions of TagSNPs were also significantly related to some of PMQ traits. In this large and comprehensive candidate gene set study, we found a modest association of genes and SNPs in the PPAR signaling pathway with PMQ. Further investigation of these gene polymorphisms jointly with fatty acid measures and other genetic factors would help us better understand the regulation mechanisms of PMQ.

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARβ and RXRβ exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRβ) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARβ, RXRβ, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRβ genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors.

Retinoid X receptor (RXR) is an important member of the nuclear receptor superfamily of ligand-activated transcription factors that are present in all major groups of metazoans. A full-length cDNA encoding RXR, an orthologue of SmRXR1 in platyhelminth Schistosoma japonicum (SjRXR1) was identified and characterized. The SjRXR1 cDNA is 2806 bp long, and contains an open reading frame encoding a 745 amino acid protein. The deduced SjRXR1 protein sequence which was aligned with RXR proteins from other species revealed a highly conserved DNA binding domain (DBD) and moderately conserved ligand binding domain (LBD). The gene structure of SjRXR1 was analyzed and showed that it consists of seven exons spanning 18.4 kbp. The relative mRNA expression of SjRXR1 was evaluated in six different S. japonicum developmental stages in the final host (days 7-42 post-infection) and showed higher expression at days 21 and 35. In an in vitro study the transcription of SjRXR1 mRNA was shown to increase almost 3-fold and the SjRXR1 protein expression was also upregulated at the 48 h time point by treating the S. japonicum with 5.0 μM 9-cis-retinoic acid (RA). Flow cytometry analysis demonstrated that the percentage of HeLa cells expressing SjRXR1LBD-Myc fusion protein is approximately 11%. Over-expression of SjRXR1LBD-Myc in HeLa cells may result in the inhibition of innate apoptosis of this cancer cell line induced by 9-cis-RA. Our studies suggested that the retinoid signaling pathways may be conserved in the platyhelminth. The full cDNA sequence of SjRXR1 reported here has been submitted to the GenBank with accession no. JX111997.

Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARβ/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket.

The objectives of the present study were to investigate the effects of acute exposure to PBDEs on retinoid signaling in fish. Zebrafish embryos (2 h post-fertilization, hpf) were exposed to DE-71 (0, 31.0, 68.7, and 227.6 mu g/L) until 120 hpf. Retinoid profiles showed the content of retinal and retinoic acid was reduced significantly. While a significant up-regulation was observed in the transcription of retinal dehydrogenase (raldh2), the transcription of retinol binding protein (rbp1a), retinol dehydrogenase (rdh1), cellular retinoic acid binding protein.(crabp1a and crabp2a) and retinoic acid receptor subunit (raraa) were down-regulated significantly, indicating disruption of retinoid signaling. However, the transcriptions of five opsin genes (zfrho, zfuv, zfred, zfblue, and zfgr1) were up-regulated. Furthermore, whole mount immunostaining and western blotting demonstrated increased rhodopsin protein expression in the exposure groups. Overall, the results indicated that acute exposure to PBDEs could disturb retinoid signaling and may impact on eye development of zebrafish larvae. (C) 2012 Elsevier B.V. All rights reserved.

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3.

Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or

There is increasing evidence of activities of chlorinated by-products of bisphenol A (BPA) on retinoic acid system. Their agonistic and antagonistic activities to human retinoid X receptor (RXR) were assessed by a two-hybrid yeast assay. Aqueous solutions of 1mg/L BPA were chlorinated by sodium hypochlorite (NaClO). It showed that chlorination of BPA increased RXRβ antagonistic activity, while no agonistic activity was detected, showing chlorine might act as a toxic potentiator rather than a toxic deactivator in RXRβ disrupting effects. BPA and its byproducts including 2,2',6,6'-tetrachlorobisphenol A (TCBPA) and 2,4,6-trichlorophenol (TCP) were quantitatively determined by gas chromatography/mass spectrometry (GC/MS). BPA rapidly degraded. With the increasing of ICC and reaction time, concentration of formed TCBPA increased initially then decreased, while concentration of formed TCP increased stably. Using the toxic equivalent (TEQ) approach, the main contributors should be mono-, di- and tri- chlorobisphenol A at initial chlorine concentration (ICC) of 1mg/L. At ICC of 2 and 5mg/L, the main contributors were TCBPA and TCP, being 57.7%-70.7% and 45.3%-59.4%. Molecular docking showed BPA chlorination by-products might have the same mode of action with BPA, forming hydrogen bond and pi-pi interaction with their OH group or hydrophobic ring.

We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and β, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.