The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Xanthohumol, a prenylated chalcone, regulates lipid metabolism by modulating the LXRα/RXR-ANGPTL3-LPL axis in hepatic cell lines and high-fat diet-fed zebrafish models.

Angiopoietin-like 3 (ANGPTL3) acts as an inhibitor of lipoprotein lipase (LPL), impeding the breakdown of triglyceride-rich lipoproteins (TGRLs) in circulation. Targeting ANGPTL3 is considered a novel strategy for improving dyslipidemia and atherosclerotic cardiovascular diseases (ASCVD). Hops (Humulus lupulus L.) contain several bioactive prenylflavonoids, including xanthohumol (Xan), isoxanthohumol (Isoxan), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN), with the potential to manage lipid metabolism. The aim of this study was to investigate the lipid-lowering effects of Xan, the effective prenylated chalcone in attenuating ANGPTL3 transcriptional activity, both in vitro using hepatic cells and in vivo using zebrafish models, along with exploring the underlying mechanisms. Xan (10 and 20 µM) significantly reduced ANGPTL3 mRNA and protein expression in HepG2 and Huh7 cells, leading to a marked decrease in secreted ANGPTL3 proteins via hepatic cells. In animal studies, orally administered Xan significantly alleviated plasma triglyceride (TG) and cholesterol levels in zebrafish fed a high-fat diet. Furthermore, it reduced hepatic ANGPTL3 protein levels and increased LPL activity in zebrafish models, indicating its potential to modulate lipid profiles in circulation. Furthermore, molecular docking results predicted that Xan exhibits a higher binding affinity to interact with liver X receptor α (LXRα) and retinoic acid X receptor (RXR) than their respective agonists, T0901317 and 9-Cis-retinoic acid (9-Cis-RA). We observed that Xan suppressed hepatic ANGPTL3 expression by antagonizing the LXRα/RXR-mediated transcription. These findings suggest that Xan ameliorates dyslipidemia by modulating the LXRα/RXR-ANGPTL3-LPL axis. Xan represents a novel potential inhibitor of ANGPTL3 for the prevention or treatment of ASCVD.

RAR Inhibitors Display Photo-Protective and Anti-Inflammatory Effects in A2E Stimulated RPE Cells In Vitro through Non-Specific Modulation of PPAR or RXR Transactivation.

N-retinylidene-N-retinylethanolamine (A2E) has been associated with age-related macular degeneration (AMD) physiopathology by inducing cell death, angiogenesis and inflammation in retinal pigmented epithelial (RPE) cells. It was previously thought that the A2E effects were solely mediated via the retinoic acid receptor (RAR)-α activation. However, this conclusion was based on experiments using the RAR "specific" antagonist RO-41-5253, which was found to also be a ligand and partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ. Moreover, we previously reported that inhibiting PPAR and retinoid X receptor (RXR) transactivation with norbixin also modulated inflammation and angiogenesis in RPE cells challenged in the presence of A2E. Here, using several RAR inhibitors, we deciphered the respective roles of RAR, PPAR and RXR transactivations in an in vitro model of AMD. We showed that BMS 195614 (a selective RAR-α antagonist) displayed photoprotective properties against toxic blue light exposure in the presence of A2E. BMS 195614 also significantly reduced the AP-1 transactivation and mRNA expression of the inflammatory interleukin (IL)-6 and vascular endothelial growth factor (VEGF) induced by A2E in RPE cells in vitro, suggesting a major role of RAR in these processes. Surprisingly, however, we showed that (1) Norbixin increased the RAR transactivation and (2) AGN 193109 (a high affinity pan-RAR antagonist) and BMS 493 (a pan-RAR inverse agonist), which are photoprotective against toxic blue light exposure in the presence of A2E, also inhibited PPARs transactivation and RXR transactivation, respectively. Therefore, in our in vitro model of AMD, several commercialized RAR inhibitors appear to be non-specific, and we propose that the phototoxicity and expression of IL-6 and VEGF induced by A2E in RPE cells operates through the activation of PPAR or RXR rather than by RAR transactivation.

Structure-Guided Design of a Highly Potent Partial RXR Agonist with Superior Physicochemical Properties.

Retinoid X receptors (RXRs, NR2B1-3) hold therapeutic potential in oncology, neurodegeneration, and metabolic diseases, but traditional RXR agonists mimicking the natural ligand 9-cis retinoic acid exhibit poor physicochemical properties, pharmacokinetics, and safety profiles. Improved RXR ligands are needed to exploit RXR modulation as a promising therapeutic concept in various indications beyond its current role in second-line cancer treatment. Here, we report the co-crystal structure of RXR in complex with a novel pyrimidine-based ligand and the structure-informed optimization of this scaffold to highly potent and highly soluble RXR agonists. Focused structure-activity relationship elucidation and rigidization resulted in a substantially optimized partial RXR agonist with low nanomolar potency, no cytotoxic activity, and very favorable physicochemical properties highlighting this promising scaffold for the development of next-generation RXR targeting drugs.

Structural Fusion of Natural and Synthetic Ligand Features Boosts RXR Agonist Potency.

The retinoid X receptors (RXRs) are ligand-activated transcription factors involved in, for example, differentiation and apoptosis regulation. Currently used reference RXR agonists suffer from insufficient specificity and poor physicochemical properties, and improved tools are needed to capture the unexplored therapeutic potential of RXR. Endogenous vitamin A-derived RXR ligands and the natural product RXR agonist valerenic acid comprise acrylic acid residues with varying substitution patterns to engage the critical ionic contact with the binding site arginine. To mimic and exploit this natural ligand motif, we probed its structural fusion with synthetic RXR modulator scaffolds, which had profound effects on agonist activity and remarkably boosted potency of an oxaprozin-derived RXR agonist chemotype. Bioisosteric replacement of the acrylic acid to overcome its pan-assay interference compounds (PAINS) character enabled the development of a highly optimized RXR agonist chemical probe.

Retinoid X Receptor agonists as selective modulators of the immune system for the treatment of cancer.

Upon heterodimerizing with other nuclear receptors, retinoid X receptors (RXR) act as ligand-dependent transcription factors, regulating transcription of critical signaling pathways that impact numerous hallmarks of cancer. By controlling both inflammation and immune responses, ligands that activate RXR can modulate the tumor microenvironment. Several small molecule agonists of these essential receptors have been synthesized. Historically, RXR agonists were tested for inhibition of growth in cancer cells, but more recent drug discovery programs screen new molecules for inhibition of inflammation or activation of immune cells. Bexarotene is the first successful example of an effective therapeutic that molecularly targets RXR; this drug was approved to treat cutaneous T cell lymphoma and is still used as a standard of care treatment for this disease. No additional RXR agonists have yet achieved FDA approval, but several promising novel compounds are being developed. In this review, we provide an overview of the multiple mechanisms by which RXR signaling regulates inflammation and tumor immunity. We also discuss the potential of RXR-dependent immune cell modulation for the treatment or prevention of cancer and concomitant challenges and opportunities.

Development of Bexarotene Analogs for Treating Cutaneous T-Cell Lymphomas.

Bexarotene, a drug approved for treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid by its ability to act as a retinoid X receptor (RXR) agonist with high specificity. Rexinoids are capable of inducing RXR homodimerization leading to the induction of apoptosis and inhibition of proliferation in human cancers. Numerous studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, many treated patients present with cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. In this study, 10 novel analogs and three standard compounds were evaluated side-by-side with bexarotene for their ability to drive RXR homodimerization and subsequent binding to the RXR response element (RXRE). In addition, these analogs were assessed for proliferation inhibition of CTCL cells, cytotoxicity, and mutagenicity. Furthermore, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Our results suggest that these new compounds may possess similar or enhanced therapeutic potential since they display enhanced RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits development of possibly more efficacious pharmaceutical drugs. Modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent compound, potentially leading to improved patient outcomes.

Direct evaluation of polarity of the ligand binding pocket in retinoid X receptor using a fluorescent solvatochromic agonist.

High selectivity of small-molecule drug candidates for their target molecule is important to minimize potential side effects. One factor that contributes to the selectivity is the internal polarity of the ligand-binding pocket (LBP) in the target molecule, but this is difficult to measure. Here, we first confirmed that the retinoid X receptor (RXR) agonist 6-(ethyl(1-isobutyl-2-oxo-4-(trifluoromethyl)-1,2-dihydroquinolin-7-yl)amino)nicotinic acid (NEt-iFQ, 1) exhibits fluorescence solvatochromism, i.e., its Stokes shift depends on the polarity of the solvent, and then we utilized this property to directly measure the internal polarity of the RXRα-LBP. The Stokes shift of 1 when bound to the RXRα-LBP corresponded to that of 1 in chloroform solution. This finding is expected to be helpful for designing RXR-selective ligands. A similar approach should be appliable to evaluate the internal polarity of the LBPs of other receptors.

Molecular interplay between ecdysone receptor and retinoid X receptor in regulating the molting of the Chinese mitten crab, Eriocheir sinensis.

Purpose: Molting is a pivotal biological process regulated by the ecdysteroid signaling pathway that requires molecular coordination of two transcription factors, Ecdysone receptor (EcR) and ultraspiracle (USP) in arthropods. However, the molecular interplay of EcR and Retinoid X receptor (RXR), the crustacean homolog of USP in the ecdysteroid signaling pathway, is not well understood. Methods: In this study, we conducted temporal and spatial expression, co-immunoprecipitation (CO-IP), and luciferase reporter assay experiments to investigate the molecular function and interplay of EcR and RXR during the molting process of the Chinese mitten crab, Eriocheir sinensis. Results: The results showed that the expression level of RXR was more stable and significantly higher than EcR during the entire molting process. However, the expression level of EcR fluctuated dynamically and increased sharply at the premolt stage. The CO-IP and luciferase reporter assay results confirmed the molecular interplay of EcR and RXR. The heterodimer complex formed by the two transcription factors significantly induced the transcription of E75, an essential gene in the ecdysteroid signaling pathway. Conclusions: Our study unveiled the diverse molecular function and molecular interplay of EcR and RXR; RXR is possibly a "constitutive-type" gene, and EcR is possibly a vital speed-limiting gene while both EcR and RXR are required to initiate the ecdysteroid signaling cascade, which may be indispensable for molting regulation in E. sinensis. The results provide a theoretical basis for the endocrine control of molting in E. sinensis and novel insights into the molecular mechanism of molting mediated by the ecdysteroid signaling pathway in crustaceans.

Vitamin A and vitamin D induced nuclear hormone receptor activation and its impact on B cell differentiation and immunoglobulin production.

Vitamin A and vitamin D metabolites are ligands to nuclear receptors - namely RAR, RXR and VDR. The activation of these receptors in human B cells impacts B cell maturation and function. In this review, we discuss how 9-cis retinoic acid (9cRA) and 1,25-dihydroxyvitamin D3 (calcitriol) individually or in conjunction, signal through their nuclear receptors and thereby impact B cell differentiation, immunoglobulin class switching to IgA at the expense of IgE, and also B cell migration and homing. Impact of the vitamin metabolites individually on B cell survival factors are well elucidated, be it the regulation of BAFF and APRIL, the induction of TGF-ß or suppression of NF-κB. Very little is known about the impact of 9cRA and calcitriol together on B cells. Recently our group revealed that 9cRA and calcitriol together in the context of the B cell differentiation, induces naïve B cell differentiation into IgA+ plasmablasts, the functional and underlying molecular regulations however require further investigation. In conclusion, the conjunctional impact of these nuclear receptor ligands on B cell functionality is important to better understand B cell dependent clinical outcomes in allergy and autoimmunity. Within this review, we hypothesize that a balance between both vitamins is of utmost importance to provide a robust humoral immune response and a better treatment of disorders characterised by dysregulated immune responses such as IgE-dependent allergy or autoimmunity such as lupus erythematosus.

Structural Modification of the Natural Product Valerenic Acid Tunes RXR Homodimer Agonism.

Retinoid X receptors (RXR) are ligand-sensing transcription factors with a unique role in nuclear receptor signaling as universal heterodimer partners. RXR modulation holds potential in cancer, neurodegeneration and metabolic diseases but adverse effects of RXR activation and lack of selective modulators prevent further exploration as therapeutic target. The natural product valerenic acid has been discovered as RXR agonist with unprecedented preference for RXR subtype and homodimer activation. To capture structural determinants of this activity profile and identify potential for optimization, we have studied effects of structural modification of the natural product on RXR modulation and identified an analogue with enhanced RXR homodimer agonism.

The novel function of bexarotene for neurological diseases.

Bexarotene, a retinoid X receptor (RXR) agonist, is approved by FDA to treat cutaneous T-cell lymphoma. However, it has also demonstrated promising therapeutic potential for neurological diseases such as stroke, traumatic brain injury, Parkinson's disease, and particularly Alzheimer's disease(AD). In AD, bexarotene inhibits the production and aggregation of amyloid ß (Aß), activates Liver X Receptor/RXR heterodimers to increase lipidated apolipoprotein E to remove Aß, mitigates the negative impact of Aß, regulates neuroinflammation, and ultimately improves cognitive function. For other neurological diseases, its mechanisms of action include inhibiting inflammatory responses, up-regulating microglial phagocytosis, and reducing misfolded protein aggregation, all of which aid in alleviating neurological damage. Here, we briefly discuss the characteristics, applications, and adverse effects of bexarotene, summarize its pharmacological mechanisms and therapeutic results in various neurological diseases, and elaborate on the problems encountered in preclinical research, with the aim of providing help for the further application of bexarotene in central nervous system diseases.

Steatosis and Metabolic Disorders Associated with Synergistic Activation of the CAR/RXR Heterodimer by Pesticides.

The nuclear receptor, constitutive androstane receptor (CAR), which forms a heterodimer with the retinoid X receptor (RXR), was initially reported as a transcription factor that regulates hepatic genes involved in detoxication and energy metabolism. Different studies have shown that CAR activation results in metabolic disorders, including non-alcoholic fatty liver disease, by activating lipogenesis in the liver. Our objective was to determine whether synergistic activations of the CAR/RXR heterodimer could occur in vivo as described in vitro by other authors, and to assess the metabolic consequences. For this purpose, six pesticides, ligands of CAR, were selected, and Tri-butyl-tin (TBT) was used as an RXR agonist. In mice, CAR's synergic activation was induced by dieldrin associated with TBT, and combined effects were induced by propiconazole, bifenox, boscalid, and bupirimate. Moreover, a steatosis, characterized by increased triglycerides, was observed when TBT was combined with dieldrin, propiconazole, bifenox, boscalid, and bupirimate. Metabolic disruption appeared in the form of increased cholesterol and lowered free fatty acid plasma levels. An in-depth analysis revealed increased expression of genes involved in lipid synthesis and lipid import. These results contribute to the growing understanding of how environmental contaminants can influence nuclear receptor activity and associated health risks.

Phase separation propensity of the intrinsically disordered AB region of human RXRß.

RXRß is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes - RXRα and RXRγ - RXRß has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRß (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid-liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR - RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR. Video Abstract.

γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice.

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

Endocytosis of LXRs: Signaling in liver and disease.

Nuclear receptors are among one of the major transcriptional factors that induces gene regulation in the nucleus. Liver X receptor (LXR) is a transcription factor which regulates essential lipid homeostasis in the body including fatty acid, cholesterol and phospholipid synthesis. Liver X receptor-retinoid X receptor (LXR-RXR) heterodimer is activated by either of the ligand binding on LXR or RXR. The promoter region of the gene which is targeted by LXR is bound to the response element of LXR. The activators bind to the heterodimer once the corepressor is dissociated. The cellular process such as endocytosis aids in intracellular trafficking and endosomal formation in transportation of molecules for essential signaling within the cell. LXR isotypes play a crucial role in maintaining lipid homeostasis by regulating the level of cholesterol. In the liver, the deficiency of LXRα can alter the normal physiological conditions depicting the symptoms of various cardiovascular and liver diseases. LXR can degrade low density lipoprotein receptors (LDLR) by the signaling of LXR-IDOL through endocytic trafficking in lipoprotein uptake. Various gene expressions associated with cholesterol level and lipid synthesis are regulated by LXR transcription factor. With its known diversified ligand binding, LXR is capable of regulating expression of various specific genes responsible for the progression of autoimmune diseases. The agonists and antagonists of LXR stand to be an important factor in transcription of the ABC family, essential for high density lipoprotein (HDL) formation. Endocytosis and signaling mechanism of the LXR family is broad and complex despite their involvement in cellular growth and proliferation. Here in this chapter, we aimed to emphasize the master regulation of LXR activation, regulators, and their implications in various metabolic activities especially in lipid homeostasis. Furthermore, we also briefed the significant role of LXR endocytosis in T cell immune regulation and a variety of human diseases including cardiovascular and neuroadaptive.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics.

Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.