Retinoid X Receptor Antagonist Research Articles

MiR-223 Plays a Key Role in Obesogen-Enhanced Adipogenesis in Mesenchymal Stem Cells and in Transgenerational Obesity.

Exposure of pregnant F0 mouse dams to the obesogen tributyltin (TBT) predisposes unexposed male descendants to obesity and diverts mesenchymal stem cells (MSCs) toward the adipocytic lineage. TBT promotes adipogenic commitment and differentiation of MSCs in vitro. To identify TBT-induced factors predisposing MSCs toward the adipocytic fate, we exposed mouse MSCs to TBT, the peroxisome proliferator activated receptor gamma (PPARγ)-selective agonist rosiglitazone, or the retinoid X receptor (RXR)-selective agonist LG-100268. Then we determined their transcriptomal profiles to determine candidate microRNAs (miR) regulating adipogenic commitment and differentiation. Of the top 10 candidate microRNAs predicted by Ingenuity Pathway Analysis, miR-21, miR-33, and miR-223 were expressed consistent with an ability to differentially regulate target genes during adipogenesis. We found that 24-hour exposure to 50nM TBT caused miR-223 levels in MSCs to increase; expression of its target genes ZEB1, NFIB, and FOXP1 was decreased. Rosiglitazone and TBT increased miR-223 levels. This induction was inhibited by the PPARγ antagonist T0070907 but not by the RXR antagonists HX531 or UVI3003, placing miR-223 downstream of PPARγ. Chromatin immunoprecipitation confirmed TBT-induced binding of PPARγ to regulatory elements in the miR-223 promoter. miR-223 levels were elevated in white adipose tissue of F2 and F3 male descendants of pregnant F0 mouse dams exposed to 50nM TBT throughout gestation. miR-223 levels were potentiated in males fed an increased fat diet. We infer that TBT induced miR-223 expression and increased adipogenesis in MSCs through the PPARγ pathway and that transgenerationally increased expression of miR-223 plays an important role in the development of obesity caused by TBT exposure.

Regulation of adipogenesis through retinoid X receptor and/or peroxisome proliferator-activated receptor by designed lignans based on natural products in 3T3-L1 cells.

We previously synthesized two retinoid X receptor (RXR) agonists, 4'-hydroxy-3'-propyl-[1,1'-biphenyl]-3-propanoic acid ethyl ester (4'OHE) and 6-hydroxy-3'-propyl-[1,1'-biphenyl]-3-propanoic acid ethyl ester (6OHE), based on the structure of magnaldehyde B, a natural product obtained from Magnolia obovata. 4'OHE and 6OHE exhibited different selectivities for peroxisome proliferator-activated receptor (PPAR)/RXR heterodimers. To examine the regulatory effects of these compounds in adipogenesis, 3T3-L1 mouse preadipocytes were treated with a differentiation cocktail with or without test compounds to induce differentiation, and subsequently treated with test compounds in insulin-containing medium every alternate day. Lipid droplets were stained with Oil Red O to examine lipid accumulation. In addition, adipogenesis-related gene expression was measured using RT-qPCR and immunoblotting. The results showed that a PPARγ agonist, 4'OHE, which exerts agonistic effects on PPARγ and RXRα, enhanced adipogenesis similar to rosiglitazone. However, unlike GW501516, a PPARδ agonist, 6OHE and its hydrolysis product (6OHA), which exert agonistic effects on PPARδ and RXRα, suppressed adipogenesis. In a manner similar to 6OHE and 6OHA, bexarotene, an RXR agonist, suppressed adipocyte differentiation, and its anti-adipogenic effect was reversed by an RXR antagonist. Furthermore, 6OHA and bexarotene inhibited the increase in Pparγ2 and Cebpa mRNA levels 2 days after the induction of differentiation. We demonstrated the adipogenic effect of 4'OHE and anti-adipogenic effects of 6OHE and 6OHA in 3T3-L1 cells. Previously, RXR agonists have been reported to positively regulate the differentiation of mesenchymal stem cells into adipocytes, but our current data showed that they inhibited the differentiation of preadipocytes, at least 3T3-L1 cells, into adipocytes.

Bexarotene enhances astrocyte phagocytosis via ABCA1-mediated pathways in a mouse model of subarachnoid hemorrhage

Background and purposeEnhancing phagocytosis can facilitate the removal of inflammatory molecules, limit the toxicity of dead cells and debris, and promote recovery after brain injury. In this study, we aimed to explore the role of bexarotene (Bex), a retinoid X receptor (RXR) agonist, in promoting astrocyte phagocytosis and neurobehavioral recovery after subarachnoid hemorrhage (SAH). MethodsMice SAH model was induced by pre-chiasmatic injection of blood. Modified Garcia score, novel object recognition, rotarod test, and Morris water maze were performed to assess neurological function. Immunofluorescence and electron microscopy were used to evaluate astrocyte phagocytosis in vivo. In addition， ABCA1/MEGF10&GULP1, the primary astrocyte phagocytosis pathway, were stimulated by Bex or suppressed by HX531 (a RXR antagonist) to evaluate their impacts on astrocyte phagocytosis and neurological recovery. ResultsAstrocytes phagocytosis of blood components were observed in mice after SAH induction, which is further increased by Bex treatment. Bex dramatically attenuated neuroinflammation, reduced brain edema, improved early neurological performance and promoted neurocognitive recovery. Meanwhile, Bex decreased neurotoxic reactive astrocytes and preserved neurogenesis after SAH. Bex increased the expression of astrocyte phagocytosis-related proteins ABCA1, MEGF10, and GULP1. Bex also increased the lysosomal processing of engulfed blood components in astrocytes. Moreover, Bex significantly promoted astrocytes to phagocytize debris in vitro by increasing the expression of ABCA1, MEGF10 and GULP1, while HX531 inhibited astrocyte phagocytosis and decreased these protein levels. ConclusionsBex enhanced astrocyte phagocytosis through the ABCA1-mediated pathways, and promoted neurobehavior recovery in mice after SAH induction.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRβ and RXRβ knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Retinoid-X receptor agonists increase thyroid hormone competence in lower jaw remodeling of pre-metamorphic Xenopus laevis tadpoles.

Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, we've used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development.

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell–cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 μM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell–cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 μM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.

A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -β/δ, -γ, and RXR antagonists and by norbixin.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9’-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -β/δ and –γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E’s deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 μM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 μM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

Retinoids promote penis development in sequentially hermaphroditic snails

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

Creation of Fluorescent RXR Antagonists Based on CBTF-EE and Application to a Fluorescence Polarization Binding Assay.

Retinoid X receptor (RXR) ligands often bind in modes in which the carboxy group forms a hydrogen bond inside the ligand-binding pocket (LBP). However, our previously reported RXR antagonist, CBTF-EE (4a), binds with its carboxy group directed outside the LBP and its alkoxy side chain located inside the LBP. Here, we examined the binding modes of 4b and 4c bearing a nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY) fluorophore, respectively, at the end of the alkoxy chain of 4a. Both compounds function as RXR antagonists. 4c, but not 4b, was available for a fluorescence polarization binding assay, indicating that rotation of BODIPY, but not NBD, is restricted in the bound state. The fluorescence findings, supported by docking simulations, suggest the fluorophores are located outside the LBP, so that the binding mode of 4b and 4c is different from that of 4a. The assay results were highly correlated with those of a [3H]9-cis-retinoic acid assay.

RXR Negatively Regulates Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells.

Ex vivo expansion of human cord blood (CB) hematopoietic stem cells (HSCs) is one approach to overcome limited numbers of HSCs in single CB units. However, there is still no worldwide acceptable HSC ex vivo expansion system. A main reason is that we still have very limited knowldege regarding mechanisms underlying maintenance and expansion of CB HSCs. Here we report that retinoid X receptor (RXR) activity is of significance for CB HSC ex vivo expansion. RXR antagonist HX531 significantly promoted ex vivo expansion of CB HSCs and progenitor cells (HPCs). RXR agonist Bexarotene notably suppressed ex vivo expansion of CB HSCs. Activation of RXR by Bexarotene significantly blocked expansion of phenotypic HSCs and HPCs and expressed increased functional HPCs as assessed by colony formation induced by UM171 and SR1. In vivo transplantation experiments in immune-deficient mice demonstrated that HX531 expanded CB HSCs possess long-term reconstituting capacities, and Bexarotene treatment inhibited expansion of functional CB HSCs. RNA-seq analysis revealed that RXR regulates expression of FBP1 (a negative regulator of glucose metabolism) and many genes involved in differentation. ECAR analysis showed that HX531 significantly promoted glycolytic activity of CB CD34+ HSCs and HPCs. Our studies suggest that RXR is a negative regulator of ex vivo expansion of CB HSCs and HPCs.

Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343.

Retinoid X receptor (RXR) modulators (rexinoids) are considered to have therapeutic potential for multiple diseases, such as Alzheimer's disease and Parkinson's disease. To overcome various disadvantages of prior screening methods, we previously developed an RXR binding assay using a fluorescent RXR ligand, CU-6PMN (4). However, this ligand binds not only at the ligand-binding domain (LBD) but also at the dimer-dimer interface of hRXRα. Here, we present a new fluorescent RXR antagonist 6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamido)phenyl)amino]nicotinic acid (NEt-C343, 7), which emits strong fluorescence only when bound to the RXR-LBD. It allows us to perform a rapid, simple, and nonhazardous binding assay that does not require bound/free separation and uses a standard plate reader. The obtained Ki values of known compounds were correlated with the Ki values obtained using the standard [3H]9cis-retinoic acid assay. This assay should be useful for drug discovery as well as for research on endocrine disruptors, functional foods, and natural products.

Discovery of a "Gatekeeper" Antagonist that Blocks Entry Pathway to Retinoid X Receptors (RXRs) without Allosteric Ligand Inhibition in Permissive RXR Heterodimers.

Retinoid X receptor (RXR) heterodimers such as PPAR/RXR, LXR/RXR, and FXR/RXR can be activated by RXR agonists alone and are therefore designated as permissive. Similarly, existing RXR antagonists show allosteric antagonism toward partner receptor agonists in these permissive RXR heterodimers. Here, we show 1-(3-(2-ethoxyethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazole-5-carboxylic acid (14, CBTF-EE) as the first RXR antagonist that does not show allosteric inhibition in permissive RXR heterodimers. This compound was designed based on the hypothesis that RXR antagonists that do not induce conformational changes of RXR would not exhibit such allosteric inhibition. CD spectra and X-ray co-crystallography of the complex of 14 and the RXR ligand binding domain (LBD) confirmed that 14 does not change the conformation of hRXR-LBD. The X-ray structure analysis revealed that 14 binds at the entrance of the ligand binding pocket (LBP), blocking access to the LBP and thus serving as a "gatekeeper".

Discovery and SAR of Natural-Product-Inspired RXR Agonists with Heterodimer Selectivity to PPARδ-RXR.

A known natural product, magnaldehyde B, was identified as an agonist of retinoid X receptor (RXR) α. Magnaldehyde B was isolated from Magnolia obovata (Magnoliaceae) and synthesized along with more potent analogs for screening of their RXRα agonistic activities. Structural optimization of magnaldehyde B resulted in the development of a candidate molecule that displayed a 440-fold increase in potency. Receptor-ligand docking simulations indicated that this molecule has the highest affinity with the ligand binding domain of RXRα among the analogs synthesized in this study. Furthermore, the selective activation of the peroxisome proliferator-activated receptor (PPAR) δ-RXR heterodimer with a stronger efficacy compared to those of PPARα-RXR and PPARγ-RXR was achieved in luciferase reporter assays using the PPAR response element driven reporter (PPRE-Luc). The PPARδ activity of the molecule was significantly inhibited by the antagonists of both RXR and PPARδ, whereas the activity of GW501516 was not affected by the RXR antagonist. Furthermore, the molecule exhibited a particularly weak PPARδ agonistic activity in reporter gene assays using the Gal4 hybrid system. The obtained data therefore suggest that the weak PPARδ agonistic activity of the optimized molecule is synergistically enhanced by its own RXR agonistic activity, indicating the potent agonistic activity of the PPARδ-RXR heterodimer.

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P.cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0±0.52, 6.7±0.37, and 9.4±0.65μM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P.cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

Assessing the endocrine disrupting potentials and genotoxicity in environmental samples from Taiwanese rivers

BackgroundSurface waters receive a variety of organic pollutants via wastewater discharge, and sediment represents a sink for hydrophobic contaminants. In this study, we used in vitro yeast-based reporter gene assays and a Bacillus subtilis Rec-assay to examine the occurrence of endocrine disrupting activities and genotoxic potentials in samples collected from three Taiwanese rivers. Levels of 51 polycyclic aromatic hydrocarbons (PAHs) in muscles of fish captured from same rivers were also analyzed to assess in vivo pollution of PAHs.ResultsAntagonist activities for androgen receptor and retinoid X receptor (RXR) were detected in river water extracts at environmentally relevant concentrations., and sediment extracts exhibited RXR agonist, RXR antagonist, and genotoxic potentials concurrently. Σ16 PAHs in fish muscles ranged from 44.9–242.4 ng g− 1 dry weight, representing 38 to 59% of the total 51 PAHs concentrations, and methylated PAHs of low molecular weight PAHs were often detected as well.ConclusionTaiwanese river sediment samples concomitantly exhibited RXR disrupting potentials and genotoxic activities, whereas RXR agonist and antagonist activities were simultaneously detected in several dry-season sediment extracts. PAH levels in fish muscles were categorized as minimally polluted by aromatic compounds, nonetheless, the presence of methylated PAHs in muscles samples may be of concern owing to the higher toxic potentials than their parent compounds.

Retinoid X receptor-mediated neuroprotection via CYP19 upregulation and subsequent increases in estradiol synthesis

Increasing evidence has shown that one of the major neurosteroids, estradiol, has potent neuroprotective actions. We have reported that estradiol synthesis was enhanced when retinoic acid was added into rat hippocampal slice culture. In this study, we investigated the effects of a potent retinoid X receptor (RXR) agonist, bexarotene, on estrogen synthesis and neuroprotective action in hippocampal slices. Treatment with bexarotene increased estradiol levels as well as estrogen-synthesizing enzymes and CYP19 expression in hippocampal slice cultures. Bexarotene significantly suppressed neuronal cell death induced by oxygen-glucose deprivation (OGD)/reoxygenation. RXR agonists other than bexarotene, such as CD3254, also suppressed neuronal cell death accompanied by OGD/reoxygenation. The RXR antagonists HX531 and UVI3003 and the CYP19 inhibitor letrozole abolished the neuroprotection elicited by bexarotene, indicating that estradiol produced by RXR stimulation protects neurons from ischemic insult. The human brain-specific CYP19 promoter had 6 RXR half sites, and 2 of 6 half sites were responsible for CYP19 expression induced by bexarotene. Bexarotene increased the expression of catalase and glutathione peroxidase 1 and inhibited lipid peroxidation elicited by OGD/reoxygenation, suggesting that the antioxidative property of estrogen contributes to RXR-mediated neuroprotection. Bexarotene also suppressed neuronal injury induced by lipopolysaccharide in the hippocampal slices. Taken together, RXR stimulation can protect neurons via enhanced synthesis of estradiol with antioxidative mechanisms. The RXR-estrogen axis might be a novel mechanism-based strategy to prevent or ameliorate ischemic and/or inflammatory neuronal disorders.

Retinoid receptor-based signaling plays a role in voltage-dependent inhibition of invertebrate voltage-gated Ca2+ channels

The retinoic acid receptor (RAR) and retinoid X receptor (RXR) mediate the cellular effects of retinoids (derivatives of vitamin A). Both RAR and RXR signaling events are implicated in hippocampal synaptic plasticity. Furthermore, retinoids can interact with calcium signaling during homeostatic plasticity. We recently provided evidence that retinoids attenuate calcium current (ICa) through neuronal voltage-gated calcium channels (VGCCs). We now examined the possibility that constitutive activity of neuronal RXR and/or RAR alters calcium influx via the VGCCs. We found that in neurons of the mollusk Lymnaea stagnalis, two different RXR antagonists (PA452 and HX531) had independent and opposing effects on ICa that were also time-dependent; whereas the RXR pan-antagonist PA452 enhanced ICa, HX531 reduced ICa Interestingly, this effect of HX531 occurred through voltage-dependent inhibition of VGCCs, a phenomenon known to influence neurotransmitter release from neurons. This inhibition appeared to be independent of G proteins and was largely restricted to Cav2 Ca2+ channels. Of note, an RAR pan-antagonist, LE540, also inhibited ICa but produced G protein-dependent, voltage-dependent inhibition of VGCCs. These findings provide evidence that retinoid receptors interact with G proteins in neurons and suggest mechanisms by which retinoids might affect synaptic calcium signaling.

Activation of retinoid X receptor by bexarotene attenuates neuroinflammation via PPAR\u03b3/SIRT6/FoxO3a pathway after subarachnoid hemorrhage in rats

BackgroundSubarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high mortality and disabilities. Retinoid X receptor (RXR) has been shown to be neuroprotective against ischemia/reperfusion injury. This study aimed to investigate the effects of the selective RXR agonist bexarotene on neuroinflammation in a rat model of SAH.MethodsTwo hundred male Sprague-Dawley rats were used. The endovascular perforation induced SAH. Bexarotene was administered intraperitoneally at 1 h after SAH induction. To investigate the underlying mechanism, the selective RXR antagonist UVI3003 and RXR siRNA or SIRT6 inhibitor OSS128167 was administered via intracerebroventricular 1 h before SAH induction. Post-SAH assessments including SAH grade, neurological score, brain water content, Western blot, and immunofluorescence were performed.ResultsThe endogenous RXR and sirtuin 6 (SIRT6) protein levels were increased after SAH. Bexarotene treatment significantly reduced brain edema and improved the short-/long-term neurological deficit after SAH. Mechanistically, bexarotene increased the levels of PPARγ and SIRT6; decreased the expression of phosphorylated FoxO3a (p-FoxO3a), IL-6, IL-1β, and TNF-a; and inhibited the microglia activation and neutrophils infiltration at 24 h after SAH. Either UVI3003, OSS128167, or RXR siRNA abolished the neuroprotective effects of bexarotene and its regulation on protein levels of PPARγ/SIRT6/p-FoxO3a after SAH.ConclusionsThe activation of RXR by bexarotene attenuated neuroinflammation and improved neurological deficits after SAH. The anti-neuroinflammatory effect was at least partially through regulating PPARγ/SIRT6/FoxO3a pathway. Bexarotene may be a promising therapeutic strategy in the management of SAH patients.

Neuroprotective effect of naturally occurring RXR agonists isolated from Sophora tonkinensis Gagnep. on amyloid-β-induced cytotoxicity in PC12 cells.

Neuronal cell death induced by amyloid-β (Aβ) oligomers is implicated in neuronal degeneration and is a leading cause of Alzheimer's disease (AD). Therefore, to identify effective therapeutic agents for AD, we investigated the neuroprotective effects of two naturally occurring retinoid X receptor (RXR) agonists (SPF1 and SPF2), isolated from the root of Sophora tonkinensis Gagnep., on the Aβ25-35-induced cytotoxicity against nerve growth factor-differentiated rat pheochromocytoma (PC12) cells. Pretreatment with SPFs significantly prevented Aβ25-35-induced apoptosis in PC12 cells, similarly to the synthetic RXR agonist bexarotene. These effects were blocked by the RXR antagonist PA452. When the effects of SPFs were studied in the presence of the liver X receptor (LXR) agonist T0901317, the protective effects of SPFs were enhanced, suggesting that RXR/LXR heterodimers may play a key role in the neuroprotective effects of SPFs. SPFs and T0901317 induced ATP-binding cassette transporter 1 (ABCA1) protein expression in PC12 cells when administered alone or in combination. Intriguingly, a functional inhibitor of ABCA1 cyclosporine A negated the neuroprotective effects of SPFs or T0901317. Taken together, these results demonstrate that the RXR agonists SPF1 and SPF2 protect PC12 cells from Aβ25-35-induced neurotoxicity in an RXR-dependent manner and that their effects are markedly enhanced by the LXR agonist T0901317, in part related to ABCA1 function. These results suggest a novel approach to the treatment or prevention of AD.