The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

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.

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.

Development of Scaled-Up Synthetic Method for Retinoid X Receptor Agonist NEt-3IB Contributing to Sustainable Development Goals.

Small-molecular drugs, which are generally inexpensive compared with biopharmaceuticals and can often be taken orally, may contribute to the Sustainable Development Goals (SDGs) adopted by the United Nations. We previously reported the retinoid X receptor (RXR) agonist 4-(ethyl(3-isobutoxy-4-isopropylphenyl)amino)benzoic acid (NEt-3IB, 1) as a small-molecular drug candidate to replace biopharmaceuticals for the treatment of inflammatory bowel disease. The previous synthetic method to 1 required a large amount of organic solvent and extensive purification. In line with the SDGs, we aimed to develop an environmentally friendly, inexpensive method for the large-scale synthesis of 1. The developed method requires only a hydrophobic ether and EtOH as reaction and extraction solvents. The product was purified by recrystallization twice to afford 99% pure 1 at 100 mmol scale in about 30% yield. The optimized process showed a 35-fold improvement of the E-factor (an index of environmental impact) compared to the original method. This work, which changes the solvent used to environmentally preferable ones based on the existing synthetic method for 1, illustrates how synthetic methods for small-molecular drugs can be adapted and improved to contribute to the SDGs.

Suppression of TLR4/MyD88/TAK1/NF-κB/COX-2 Signaling Pathway in the Central Nervous System by Bexarotene, a Selective RXR Agonist, Prevents Hyperalgesia in the Lipopolysaccharide-Induced Pain Mouse Model.

A selective RXR agonist, bexarotene, has been shown to have anti-inflammatory, anti-nociceptive, and neuroprotective effects in several models of numerous neurological diseases characterized by systemic inflammation. The mechanisms underlying these effects remains unknown. To elucidate these mechanisms, we investigated whether the TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in the CNS mediates the effect of bexarotene to prevent hyperalgesia in the LPS-induced inflammatory pain mouse model. The reaction time to thermal stimuli within 30 s was evaluated by the hot plate test in male mice treated with saline, LPS (10 mg/kg), DMSO, and/or bexarotene (0.1, 1, 3, or 10 mg/kg) after 6 h. The latency to the thermal stimulus (18.11 ± 1.36 s) in the LPS-treated mice was significantly decreased by 30% compared with saline-treated mice (25.84 ± 1.99 s). Treatment with bexarotene only at a dose of 10 mg/kg showed a significant increase in the latency by 22.49 ± 1.00 s compared with LPS-treated mice. Bexarotene also prevented the reduction in RXRα protein expression associated with a rise in the expression of TLR4, MyD88, phosphorylated TAK1, NF-κB p65, phosphorylated NF-κB p65, COX-2, and IL-1ß proteins, in addition to COX-2 activity and levels of PGE2 and IL-1ß in the brains and spinal cords of the LPS-treated animals. Likely, decreased activity of TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in addition to increased pro-inflammatory cytokine formation in the CNS of mice participates in the protective effect of bexarotene against hyperalgesia induced by LPS.

DHA and Its Metabolites Have a Protective Role against Methylmercury-Induced Neurotoxicity in Mouse Primary Neuron and SH-SY5Y Cells.

The consumption of fish now involves a risk of methylmercury (MeHg) exposure but also provides the benefit of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) such as docosahexaenoic acid (DHA). Some epidemiological studies have suggested that the intake of DHA can alleviate the neurotoxicity of MeHg, but the underlying mechanism is not known. Herein, we observed that pretreatment with 0.1-1 µM DHA suppressed MeHg-induced cytotoxicity in human neuroblastoma (SH-SY5Y) cells and mouse primary neuronal cells. These effects of DHA were canceled in the presence of the retinoid X receptor (RXR) antagonist UVI3003. An RXR agonist, bexarotene, suppressed the cytotoxicity of MeHg. DHA also suppressed the MeHg-induced production of reactive oxygen species (ROS) via an induction of antioxidant genes (catalase and SOD1). Pretreatment with DHA did not change the incorporation of MeHg. We showed previously that in the brain, the intake of DHA increased the level of 19,20-DHDP, which is the metabolite produced by cytochrome P450 and soluble epoxide hydrolase from DHA. In the present study, we observed that 19,20-DHDP also suppressed neurotoxicity from MeHg. These results indicate that DHA and its metabolites have a protective role in MeHg-induced neurotoxicity.

A partial agonist for retinoid X receptor mitigates experimental colitis.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is an intractable disease of the gastrointestinal tract. Multiple environmental factors, including food ingredients, have been implicated in the development of these diseases. For example, animal fat-rich diets are predisposing factors for ulcerative colitis, whereas n-3 unsaturated fatty acids such as docosahexaenoic acid (DHA) show protective effects in experimental colitis and are negatively correlated with the incidence of ulcerative colitis and Crohn's disease. Given that DHA exhibits agonistic activity on retinoid X receptor (RXR), activation of RXR could be a therapeutic strategy for IBD. However, conventional full RXR agonists are known to show considerable adverse effects. We therefore took advantage of a partial RXR agonist, CBt-PMN, to minimize the adverse effects, and evaluated its efficacy in dextran sodium sulfate-induced colitis. Administration of CBt-PMN efficiently ameliorated the symptoms of colitis. This effect was attributed to the down-regulation of pro-inflammatory cytokines such as Tnf and Il6 in colon-infiltrating monocytes. Down-regulation of pro-inflammatory cytokines by CBt-PMN was also evident in lipopolysaccharide-stimulated bone marrow-derived macrophages (BMDMs). Among many RXR-associated nuclear receptors, activation of peroxisome proliferator-activated receptor δ (PPARδ) and nuclear hormone receptor 77 (Nur77) suppressed cytokine production by BMDMs. These observations suggest that the activation of PPARδ/RXR and Nur77/RXR heterodimers by CBt-PMN through the permissive mechanism is responsible for diminishing the monocyte-mediated inflammatory response in the gut. Our data highlight the importance of RXR activation in the regulation of colitis.

Retinoid X receptor agonists attenuates cardiomyopathy in streptozotocin-induced type 1 diabetes through LKB1-dependent anti-fibrosis effects.

Diabetic cardiac fibrosis increases ventricular stiffness and facilitates the occurrence of diastolic dysfunction. Retinoid X receptor (RXR) plays an important role in cardiac development and has been implicated in cardiovascular diseases. In the present study, we investigated the effects of RXR agonist treatment on streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM) and the underlying mechanism. Sprague-Dawley (SD) rats induced by STZ injection were treated with either RXR agonist bexarotene (Bex) or vehicle alone. Echocardiography was performed to determine cardiac structure and function. Cardiac fibroblasts (CFs) were treated with high glucose (HG) with or without the indicated concentration of Bex or the RXR ligand 9-cis-retinoic acid (9-cis-RA). The protein abundance levels were measured along with collagen, body weight (BW), blood biochemical indexes and transforming growth factor-ß (TGF-ß) levels. The effects of RXRα down-regulation by RXRα small interfering RNA (siRNA) were examined. The results showed that bexarotene treatment resulted in amelioration of left ventricular dysfunction by inhibiting cardiomyocyte apoptosis and myocardial fibrosis. Immunoblot with heart tissue homogenates from diabetic rats revealed that bexarotene activated liver kinase B1 (LKB1) signaling and inhibited p70 ribosomal protein S6 kinase (p70S6K). The increased collagen levels in the heart tissues of DCM rats were reduced by bexarotene treatment. Treatment of CFs with HG resulted in significantly reduced LKB1 activity and increased p70S6K activity. RXRα mediated the antagonism of 9-cis-RA on HG-induced LKB1/p70S6K activation changes in vitro. Our findings suggest that RXR agonist ameliorates STZ-induced DCM by inhibiting myocardial fibrosis via modulation of the LKB1/p70S6K signaling pathway. RXR agonists may serve as novel therapeutic agents for the treatment of DCM.

Combinations of LXR and RXR agonists induce triglyceride accumulation in human HepaRG cells in a synergistic manner.

Activation of nuclear receptors (NR), for example the retinoid-X-receptors (RXR) or the liver-X-receptors (LXR), plays a crucial role as the molecular initiating event in the adverse outcome pathway for liver steatosis. The downstream biological consequences of NR interactions are still not fully understood, especially with multi-receptor-activating compounds and their mixtures. While the default assumption for mixture risk assessment is dose addition, the potential of combinations of synthetic RXR agonists to exert synergistic effects has been shown in the context of NR activation studies. The fact that RXR and LXR are heterodimerization partners raises the question whether combinations of LXR and RXR agonists may cause synergistic effects. Compounds with defined properties were chosen to examine their interactions regarding the activation of RXR and LXR, as well as the steatosis-related key events target gene activation and triglyceride accumulation, using the human HepaRG liver cell model. Synergistic effects were determined for cellular triglyceride accumulation, especially at high compound concentrations, as evaluated using five different mathematical models. Altered LXRα activation in the presence of RXR agonists was observed, and synergistic effects on LXR target genes were identified as a presumably underlying mechanism of the observed synergistic effect. These findings challenge the general validity of dose addition as the default assumption for mixture effects, and point toward the need for a mode of action-based risk assessment for chemical mixtures.

A review of the molecular design and biological activities of RXR agonists.

An attractive approach to combat disease is to target theregulation of cell function. At the heart of this task are nuclear receptors (NRs); which control functions such as gene transcription. Arguably, the key player in this regulatory machinery is the retinoid X receptor (RXR). This NR associates with a third of the NRs found in humans. Scientists have hypothesized that controlling the activity of RXR is an attractive approach to control cellular functions that modulate diseases such as cancer, diabetes, Alzheimer's disease and Parkinson's disease. In this review, we will describe the key features of the RXR, present a historic perspective of the first RXR agonists, and discuss various templates that have been reported to activate RXR with a focus on their molecular structure, biological activity, and limitations. Finally, we will present an outlook of the field and future directions and considerations to synthesize or modulate RXR agonists to make these compounds a clinical reality.

Activation of retinoid X receptor by bexarotene attenuates neuroinflammation via PPARγ/SIRT6/FoxO3a pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid 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. METHODS: Two 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. RESULTS: The 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-1ß, 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. CONCLUSIONS: The 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.

A retinoid X receptor partial agonist attenuates pulmonary emphysema and airway inflammation.

BACKGROUND: Retinoid X receptors (RXRs) are members of the nuclear receptor (NR) superfamily that mediate signalling by 9-cis retinoic acid, a vitamin A derivative. RXRs play key roles not only as homodimers but also as heterodimeric partners, e.g., for retinoic acid receptors, vitamin D receptors, and peroxisome proliferator-activated receptors. The NR family may also play important roles in the development of emphysema. However, the role of RXRs in the pathogenesis of emphysema is not well defined. METHODS: We developed a novel RXR partial agonist (NEt-4IB) and investigated its effect and mechanism compared to a full agonist (bexarotene) in a murine model of emphysema. For emphysema induction, BALB/c mice received intraperitoneal cigarette smoke extract (CSE) or intratracheal porcine pancreas elastase (PPE). Treatment with RXR agonists was initiated before or after emphysema induction. RESULTS: Treatment with NEt-4IB significantly suppressed the increase in static lung compliance and emphysematous changes in CSE-induced emphysema and PPE-induced established and progressive emphysema. NEt-4IB significantly suppressed PPE-induced neutrophilic airway inflammation and the levels of keratinocyte chemoattractant (KC), C-X-C motif ligand5 (CXCL5), interferon (IFN)-γ and IL-17. NEt-4IB also improved the matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1) imbalance and the reduced anti-oxidant activity in bronchoalveolar lavage (BAL) fluid. NEt-4IB suppressed PPE-induced vascular endothelial growth factor (VEGF) expression in the airway. Treatment with NEt-4IB and bexarotene significantly suppressed the increase in static lung compliance and emphysematous changes. However, adverse effects of RXR agonists, including hypertriglyceridemia and hepatomegaly, were observed in bexarotene-treated mice but not in NEt-4IB-treated mice. CONCLUSION: These data suggest that RXRs play crucial roles in emphysema and airway inflammation, and novel partial RXR agonists could be potential therapeutic strategies for the treatment of PPE- and CSE-induced emphysema.

Insights into interplay between rexinoid signaling and myogenic regulatory factor-associated chromatin state in myogenic differentiation.

While skeletal myogenesis is tightly coordinated by myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Here, we examine the genome-wide impact of rexinoids on myogenic differentiation through integral RNA-seq and ChIP-seq analyses. We found that bexarotene promotes myoblast differentiation through the coordination of exit from the cell cycle and the activation of muscle-related genes. We uncovered a new mechanism of rexinoid action which is mediated by the nuclear receptor and largely reconciled through a direct regulation of MyoD gene expression. In addition, we determined a rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated to MyoD and myogenin. Thus, we provide novel molecular insights into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation.

Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae.

Ampullariidae are unique among gastropods in that females normally show a primordium of the copulatory apparatus (CApp). The aims of this study were (a) to quantitatively evaluate the development and growth of the female CApp with age; (b) to compare the effects of RXR and PPARγ agonists in adult females of known age and (c) to explore the effect of masculinizing RXR agonists on the expression of RXR in the CApp. It was found that the CApp grows and develops with age. A significant increase in penile sheath length (PsL) and also in a developmental index (DI) was observed in 7-8 months old females, as compared with 4-5 months old ones. A reported endogenous agonist of RXR, 9-cis retinoic acid (9cis-RA), as well as two organotin compounds, tributyltin (TBT) and triphenyltin (TPT) which have been also reported to bind to RXR, were injected and its masculinizing effects were measured. Also, the effect of a PPARγ agonist, rosiglitazone, was studied. All studied RXR agonists, but not the PPARγ agonist, were effective in increasing PsL, penile length (PL) and DI. Finally, the expression of the RXR in the CApp was studied (Western blot) in control, TBT, TPT, and 9cis-RA treated females. A significantly increased expression of RXR was only observed after 9cis-RA treatment. It is concluded that (a) development and growth of the CApp is significantly affected by female age; (b) reported RXR agonists, but not a PPARγ agonist, cause female masculinization of young females. An appraisal of previous studies of female masculinization in the Ampullariidae has also been made and it is emphasized that the masculinizing effect of aging should be considered, particularly when interpreting field data.

Exposure to the RXR Agonist SR11237 in Early Life Causes Disturbed Skeletal Morphogenesis in a Rat Model.

Longitudinal bone growth occurs through endochondral ossification (EO), controlled by various signaling molecules. Retinoid X Receptor (RXR) is a nuclear receptor with important roles in cell death, development, and metabolism. However, little is known about its role in EO. In this study, the agonist SR11237 was used to evaluate RXR activation in EO. Rats given SR11237 from post-natal day 5 to post-natal day 15 were harvested for micro-computed tomography (microCT) scanning and histology. In parallel, newborn CD1 mouse tibiae were cultured with increasing concentrations of SR11237 for histological and whole-mount evaluation. RXR agonist-treated rats had shorter long bones than the controls and developed dysmorphia of the growth plate. Cells invading the calcified and dysmorphic growth plate appeared pre-hypertrophic in size and shape, in correspondence with p57 immunostaining. Additionally, SOX9-positive cells were found surrounding the calcified tissue. The epiphysis of SR11237-treated bones showed increased TRAP staining and additional TUNEL staining at the osteo-chondral junction. MicroCT revealed morphological disorganization in the long bones of the treated animals. This study suggests that stimulation of RXR causes irregular ossification, premature closure of the growth plate, and disrupted long bone growth in rodent models.

Retinoic Acid Increases Fatty Acid Oxidation and Irisin Expression in Skeletal Muscle Cells and Impacts Irisin In Vivo.

BACKGROUND/AIMS: All-trans retinoic acid (ATRA) has protective effects against obesity and metabolic syndrome. We here aimed to gain further insight into the interaction of ATRA with skeletal muscle metabolism and secretory activity as important players in metabolic health. METHODS: Cultured murine C2C12 myocytes were used to study direct effects of ATRA on cellular fatty acid oxidation (FAO) rate (using radioactively-labelled palmitate), glucose uptake (using radioactively-labelled 2-deoxy-D-glucose), triacylglycerol levels (by an enzymatic method), and the expression of genes related to FAO and glucose utilization (by RT-real time PCR). We also studied selected myokine production (using ELISA and immunohistochemistry) in ATRA-treated myocytes and intact mice. RESULTS: Exposure of C2C12 myocytes to ATRA led to increased fatty acid consumption and decreased cellular triacylglycerol levels without affecting glucose uptake, and induced the expression of the myokine irisin at the mRNA and secreted protein level in a dose-response manner. ATRA stimulatory effects on FAO-related genes and the Fndc5 gene (encoding irisin) were reproduced by agonists of peroxisome proliferator-activated receptor ß/δ and retinoid X receptors, but not of retinoic acid receptors, and were partially blocked by an AMP-dependent protein kinase inhibitor. Circulating irisin levels were increased by 5-fold in ATRA-treated mice, linked to increased Fndc5 transcription in liver and adipose tissues, rather than skeletal muscle. Immunohistochemistry analysis of FNDC5 suggested that ATRA treatment enhances the release of FNDC5/irisin from skeletal muscle and the liver and its accumulation in interscapular brown and inguinal white adipose depots. CONCLUSION: These results provide new mechanistic insights on how ATRA globally stimulates FAO and enhances irisin secretion, thereby contributing to leaning effects and improved metabolic status.

Nuclear receptor agonist-driven modification of inflammation and amyloid pathology enhances and sustains cognitive improvements in a mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a highly prevalent neurodegenerative disorder characterized by pathological hallmarks of beta-amyloid plaque deposits, tau pathology, inflammation, and cognitive decline. Treatment remains a clinical obstacle due to lack of effective therapeutics. Agonists targeting nuclear receptors, such as bexarotene, reversed cognitive deficits regardless of treatment duration and age in murine models of AD. While bexarotene demonstrated marked efficacy in decreasing plaque levels following short-term treatment, prolonged treatment did not modulate plaque burden. This suggested that plaques might reform in mice treated chronically with bexarotene and that cessation of bexarotene treatment before plaques reform might alter amyloid pathology, inflammation, and cognition in AD mice. METHODS: We utilized one-year-old APP/PS1 mice that were divided into two groups. We treated one group of mice for 2 weeks with bexarotene. The other group of mice was treated for 2 weeks with bexarotene followed by withdrawal of drug treatment for an additional 2 weeks. Cognition was evaluated using the novel-object recognition test either at the end of bexarotene treatment or the end of the withdrawal period. We then analyzed amyloid pathology and microgliosis at the conclusion of the study in both groups. RESULTS: Bexarotene treatment enhanced cognition in APP/PS1 mice similar to previous findings. Strikingly, we observed sustained cognitive improvements in mice in which bexarotene treatment was discontinued for 2 weeks. We observed a sustained reduction in microgliosis and plaque burden following drug withdrawal exclusively in the hippocampus. CONCLUSIONS: Our findings demonstrate that bexarotene selectively modifies aspects of neuroinflammation in a region-specific manner to reverse hippocampal-dependent cognitive deficits in AD mice and may provide insight to inform future studies with nuclear receptor agonists.

HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

UAB30, a novel RXR agonist, decreases tumorigenesis and leptomeningeal disease in group 3 medulloblastoma patient-derived xenografts.

BACKGROUND: Group 3 tumors account for approximately 25-30% of medulloblastomas and have the worst prognosis. UAB30 is a novel synthetic rexinoid shown to have limited toxicities in humans and significant efficacy in the pediatric neuroectodermal tumor, neuroblastoma. We hypothesized that treatment with UAB30 would decrease tumorigenicity in medulloblastoma patient-derived xenografts (PDXs). METHODS: Three group 3 medulloblastoma PDXs (D341, D384 and D425) were utilized. Cell viability, proliferation, migration and invasion assays were performed after treatment with UAB30 or 13-cis-retinoic acid (RA). Cell cycle analysis was completed using flow cytometry. A flank model, a cerebellar model, and a model of leptomeningeal metastasis using human medulloblastoma PDX cells was used to assess the in vivo effects of UAB30 and RA. RESULTS: UAB30 treatment led to cell differentiation and decreased medulloblastoma PDX cell viability, proliferation, migration and invasion and G1 cell cycle arrest in all three PDXs similar to RA. UAB30 and RA treatment of mice bearing medulloblastoma PDX tumors resulted in a significant decrease in tumor growth and metastasis compared to vehicle treated animals. CONCLUSIONS: UAB30 decreased viability, proliferation, and motility in group 3 medulloblastoma PDX cells and significantly decreased tumor growth in vivo in a fashion similar to RA, suggesting that further investigations into the potential therapeutic application of UAB30 for medulloblastoma are warranted.