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Retinitis Pigmentosa is a leading cause of severe vision loss. Retinitis Pigmentosa can present with a broad range of phenotypes impacted by disease age of onset, severity, and progression. This variation is influenced both by different gene mutations as well as unique variants within the same gene. Mutations in the nuclear hormone receptor 2 family e, member 3 are associated with several forms of retinal degeneration, including Retinitis Pigmentosa. In our previous studies we demonstrated that subretinal administration of one Nr2e3 dose attenuated retinal degeneration in rd7 mice for at least 3 months. Here we expand the studies to evaluate the efficacy and longitudinal impact of the NR2E3 therapeutic by examining three different doses administered at early or intermediate stages of retinal degeneration in the rd7 mice. Our study revealed retinal morphology was significantly improved 6 months post for all doses in the early-stage treatment groups and for the low and mid doses in the intermediate stage treatment groups. Similarly, photoreceptor function was significantly improved in the early stage for all doses and intermediate stage low and mid dose groups 6 months post treatment. This study demonstrated efficacy in multiple doses of NR2E3 therapy., (© 2024. The Author(s).)

Addiction is associated with neuroadaptive changes in the brain. In the present paper, we used a model of methamphetamine self-administration during which we used footshocks to divide rats into animals that continue to press a lever to get methamphetamine (shock-resistant) and those that significantly reduce pressing the lever (shock-sensitive) despite the shocks. We trained male Sprague-Dawley rats to self-administer methamphetamine (0.1 mg/kg/infusion) for 9 hours daily for 20 days. Control group self-administered saline. Subsequently, methamphetamine self-administration rats were punished by mild electric footshocks for 10 days with gradual increases in shock intensity. Two hours after stopping behavioral experiments, we euthanized rats and isolated nucleus accumbens (NAc) samples. Affymetrix Array experiments revealed 24 differentially expressed genes between the shock-resistant and shock-sensitive rats, with 15 up- and 9 downregulated transcripts. Ingenuity pathway analysis showed that these transcripts belong to classes of genes involved in nervous system function, behavior, and disorders of the basal ganglia. These genes included prodynorphin (PDYN) and proenkephalin (PENK), among others. Because PDYN and PENK are expressed in dopamine D1- and D2-containing NAc neurons, respectively, these findings suggest that mechanisms, which impact both cell types may play a role in the regulation of compulsive methamphetamine taking by rats.

Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39(-/-)) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39(-/-) mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr(-/-)T39(-/-)) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXRα-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.

Liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. Under conditions of excess cholesterol, LXR activation induces the expression of several genes involved in cholesterol efflux, facilitates cholesterol esterification by promoting fatty acid synthesis, and inhibits cholesterol uptake by the low-density lipoprotein receptor. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways are incompletely understood. Here we show that ligand activation of LXRs in mouse liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as a mediator of this effect. Hepatic LeXis expression is robustly induced in response to a Western diet (high in fat and cholesterol) or to pharmacological LXR activation. Raising or lowering LeXis levels in the liver affects the expression of genes involved in cholesterol biosynthesis and alters the cholesterol levels in the liver and plasma. LeXis interacts with and affects the DNA interactions of RALY, a heterogeneous ribonucleoprotein that acts as a transcriptional cofactor for cholesterol biosynthetic genes in the mouse liver. These findings outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms that coordinate sterol homeostasis.

Nuclear receptor subfamily 4 group A member 2 (NR4A2) is an orphan nuclear receptor that is over-expressed in cancer and promotes cell proliferation, migration, transformation, and chemoresistance. Increased expression and function of NR4A2 have been attributed to various signaling pathways, but little is known about microRNA (miRNA) regulation of NR4A2 in cancer. To investigate the posttranscriptional regulation of NR4A2, we used a 3' untranslated region (UTR) reporter screen and identified miR-34 as a putative regulator of NR4A2. By using computer predictions, we identified and confirmed an miRNA recognition element in the 3' UTR of NR4A2 that was responsible for miR-34-mediated suppression. We next demonstrated that overexpression of exogenous miR-34 or activation of the p53 pathway, which regulates endogenous miR-34 expression, decreased NR4A2 expression. Consistent with previous reports, overexpression of NR4A2 blocked the induction of p53 target genes, including mir-34a. This was a phenotypic effect, as NR4A2 overexpression could rescue cells from p53-induced inhibition of proliferation. In summary, our results are the first characterization of a cancer-related miRNA capable of regulating NR4A2 and suggest a network and possible feedback mechanism involving p53, miR-34, and NR4A2.

The expression of the epidermal growth factor receptor (EGFR) is highly regulated in normal cells, whereas some cancer cells have high constitutive levels. Understanding naturally-occurring ways of downregulating EGFR in cancer cells was investigated. Phosphatidic acid (PA) or Nuclear Receptors (NR) PPARα/RXRα/LXRα, enhance EGFR expression, mediated by the promoter region -856(A) to -226(T). Unexpectedly, the combination of NRs and PA caused repression. PA induces a conformational change in the nuclear receptor PPARα (increase of alpha-helices at the expense of decreasing beta-sheets), as evidenced by circular dichroism. This represses the naturally-enhancing capability of PPARα on EGFR transcription. PPARα-overexpressing cells in the presence of PA > 300 nM or the enzyme that produces it, phospholipase D (PLD), downregulate EGFR expression. The reasons are two-fold. First, PA displaces PPARα binding to the EGFR promoter at those concentrations. Second, NR heterodimer-dependent promoter activity is weakened in the presence of PA in vivo. Since other genes considered (β-catenin, cyclin D3, PLD2 and ACOX-1) are also downregulated with a PA + PPARα combination, the transrepression appears to be a global phenomenon. Lastly, the reported effect is greater in MCF-7 than in MDA-MB-231 breast cancer cells, which could provide a novel basis for regulating excessive expression of EGFR in luminal cancer cells.

Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.

Emerging evidence has linked photoreceptor cell-specific nuclear receptor (PNR/NR2E3), an orphan nuclear hormone receptor, to human breast cancer. PNR was shown to be a transcriptional activator of estrogen receptor-α (ERα) in ERα-positive breast cancer cell lines and high-level expression of PNR correlates with favorable response of ERα-positive breast cancer patients to tamoxifen. Interestingly, gene expression microarray study shows that PNR regulates distinct genes from those regulated by ERα, suggesting that PNR could have ERα-independent functions. Herein, we investigated the function of PNR in ERα-negative breast cancer cells. Our results showed that PNR-induced cell migration and metastasis of ERα-negative breast cancer cells both in vitro and in vivo, and the effect was attributed to the upregulation of interleukin (IL)-13Rα2, a high-affinity receptor for IL-13 that regulates tumor growth, invasion and metastasis of various human cancers. Mechanistically, PNR activated transcription of IL-13Rα2 through direct recruitment to IL-13Rα2 promoter. Upon stimulation with IL-13, IL-13Rα2 increased the extracellular signal-regulated kinases 1 and 2 phosphorylation, which led to breast cancer migration and metastasis. The IL-13 triggered signal cascade was specific to IL-13Rα2, as the closely related IL-13Rα1 was not regulated by PNR. IL-13Rα2 is a novel tumor antigen that is overexpressed in a variety of solid tumor types. This study presents the first evidence that PNR could promote ERα-negative breast cancer metastasis through activation of IL-13Rα2-mediated signaling pathway.

Metformin is a first-line drug for treating type 2 diabetes. Although metformin is known to phosphorylate AMP-activated protein kinase (AMPK), it is unclear how the glucose-lowering effect of metformin is related to AMPK activation. The aim of this study was to identify the urinary endogenous metabolites affected by metformin and to identify the novel underlying molecular mechanisms related to its anti-diabetic effect. Fourteen healthy male subjects were orally administered metformin (1000 mg) once. First morning urine samples were taken before and after administration to obtain metabolomic data. We then further investigated the anti-diabetic mechanism of metformin in vitro and in vivo. The fluctuation of the metabolite cortisol indicated that the neuroendocrine system was involved in the anti-diabetic effect of metformin. Actually we found that metformin induced AMPK/liver X receptor α (LXRα) phosphorylation, followed by pro-opiomelanocortin (POMC) suppression in rat pituitary cells. We confirmed this result by administering metformin in an animal study. Given that cortisol stimulates gluconeogenesis, we propose the anti-hyperglycemic effect of metformin is attributed to reduced POMC/adrenocorticotropic hormone (ACTH)/cortisol levels following AMPK/LXRα phosphorylation in the pituitaries.

Nuclear receptors (NRs) are ligand-regulated transcription factors that regulate metabolism, development and immunity. The NR superfamily is one of the major classes of drug targets for human diseases. Retinoic acid receptor-related orphan receptor (ROR) α, β and γ belong to the NR superfamily, and these receptors are still considered as 'orphan' receptors because the identification of their endogenous ligands has been controversial. Recent studies have demonstrated that these receptors are regulated by synthetic ligands, thus emerge as important drug targets for the treatment of multiple sclerosis, rheumatoid arthritis, psoriasis, etc. Studying the structural basis and ligand development of RORs will pave the way for a better understanding of the roles of these receptors in human diseases. Here, we review the structural basis, disease relevance, strategies for ligand identification, and current status of development of therapeutic ligands for RORs.

Liver X receptors (LXRs) have been proposed to have some anticancer properties, through molecular mechanisms that remain elusive. Here we report for the first time that LXR ligands induce caspase-1-dependent cell death of colon cancer cells. Caspase-1 activation requires Nod-like-receptor pyrin domain containing 3 (NLRP3) inflammasome and ATP-mediated P2 × 7 receptor activation. Surprisingly, LXRβ is mainly located in the cytoplasm and has a non-genomic role by interacting with pannexin 1 leading to ATP secretion. Finally, LXR ligands have an antitumoral effect in a mouse colon cancer model, dependent on the presence of LXRβ, pannexin 1, NLRP3 and caspase-1 within the tumor cells. Our results demonstrate that LXRβ, through pannexin 1 interaction, can specifically induce caspase-1-dependent colon cancer cell death by pyroptosis.

Liver X receptors (LXRs), including LXRα and LXRβ isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expression of cyclin D1 and cyclin B1, and thus suppresses the proliferation of multiple cancer cells, but the relevant mechanism is not well known. Forkhead box M1 (FOXM1) is a proliferation-specific member of forkhead box family, which is highly expressed in proliferating normal cells and numerous cancer cells. FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the enhancement of cell cycle progression and cell proliferation. However, it is unclear whether LXRs are involved in the regulation of FOXM1. In this study, we demonstrated that specific LXRs agonists downregulated expression of FOXM1, cyclin D1 and cyclin B1 in hepatocellular carcinoma (HCC) cells, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly alleviated LXRs activation-mediated cell cycle arrest and cell growth suppression. Reporter assays showed that the activation of LXRs significantly reduced the transcriptional activity of FOXM1 promoter. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that LXRα but not LXRβ could bind to an inverted repeat IR2 (-52CCGTCAcgTGACCT-39) in the promoter region of FOXM1 gene. Moreover, the xenograft tumor growth and the corresponding FOXM1 expression in nude mice were dramatically repressed by LXRs agonists. Taken together, we conclude that LXRα but not LXRβ functions as a transcriptional repressor for FOXM1 expression. The pathway 'LXRα-FOXM1-cyclin D1/cyclin B1' is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for HCC treatment.

Hepatic steatosis is common in obese individuals with hyperinsulinemia and is an important hepatic manifestation of metabolic syndrome. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces transcription of SREBP-1c via activation of liver X receptor (LXR) and specificity protein 1 (Sp1). Cilostazol is an antiplatelet agent that prevents atherosclerosis and decreases serum triglyceride levels. However, little is known about the effects of cilostazol on hepatic lipogenesis. Here, we examined the role of cilostazol in the regulation of SREBP-1c transcription in the liver. The effects of cilostazol on the expression of SREBP-1c and its target genes in response to insulin or an LXR agonist (T0901317) were examined using real-time RT-PCR and western blot analysis on cultured hepatocytes. To investigate the effect of cilostazol on SREBP-1c at the transcriptional level, transient transfection reporter assays and electrophoretic mobility shift assays (EMSAs) were performed. Cilostazol inhibited insulin-induced and LXR-agonist-induced expression of SREBP-1c and its downstream targets, acetyl-CoA carboxylase and fatty acid synthase, in cultured hepatocytes. Cilostazol also inhibited activation of the SREBP-1c promoter by insulin, T0901317 and Sp1 in a luciferase reporter assay. EMSA analysis showed that cilostazol inhibits SREBP-1c expression by repressing the binding of LXR and Sp1 to the promoter region. These results indicate that cilostazol inhibits insulin-induced hepatic SREBP-1c expression via the inhibition of LXR and Sp1 activity and that cilostazol is a negative regulator of hepatic lipogenesis.

Using a combination of N-ethyl-N-nitrosourea-mediated mutagenesis and metabolomics-guided screening, we identified mice with elevated blood levels of short-chain C4-acylcarnitine and increased urine isobutyryl-glycine. Genome-wide homozygosity screening, followed by fine mapping, located the disease gene to 15-25 Mb of mouse chromosome 9 where a candidate gene, Acad8, encoding mitochondrial isobutyryl-CoA dehydrogenase was located. Genomic DNA sequencing revealed a single-nucleotide mutation at -17 of the first intron of Acad8 in affected mice. cDNA sequencing revealed an intronic 28-bp insertion at the site of the mutation, which caused a frame shift with a premature stop codon. In vitro splicing assay confirmed that the mutation was sufficient to activate an upstream, aberrant 3' splice site. There was a reduction in the expression of Acad8 at both the mRNA and protein levels. The mutant mice grew normally but demonstrated cold intolerance at young age with a progressive hepatic steatosis. Homozygous mutant mice hepatocytes had abnormal mitochondria with crystalline inclusions, suggestive of mitochondriopathy. This mouse model of isobutyryl-CoA dehydrogenase deficiency could provide us a better understanding of the possible role of IBD deficiency in mitochondriopathy and fatty liver.

Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A-actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis.

The nuclear receptors, steroid and xenobiotic receptor (SXR) and constitutive androstane receptor (CAR) play important functions in mediating lipid and drug metabolism in the liver. The present study demonstrates modulatory actions of estrogen in transactivations of SXR-mediated liver X receptor response element (LXRE) and CAR-mediated phenobarbital response element (PBRU). When human estrogen receptor (hERα) and SXR were exogenously expressed, treatment with either rifampicin or corticosterone promoted significantly the SXR-mediated transactivation of LXRE reporter gene in HepG2. However, combined treatment with estrogen plus either rifampicin or corticosterone resulted in less than 50% of the mean values of the transactivation by rifampicin or corticosterone alone. Thus, it is suggested that estrogen may repress the SXR-mediated transactivation of LXRE via functional cross-talk between ER and SXR. The CAR-mediated transactivation of PBRU was stimulated by hERa in the absence of estrogen. However, the potentiation by CAR agonist, TCPOBOP, was significantly repressed by moxestrol in the presence of ER. Thus, ER may play both stimulatory and inhibitory roles in modulating CAR-mediated transactivation of PBRU depending on the presence of their ligands. In summary, this study demonstrates that estrogen modulates transcriptional activity of SXR and CAR in mediating transactivation of LXRE and PBRU, respectively, of the nuclear receptor target genes through functional cross-talk between ER and the corresponding nuclear receptors.

Aim: To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis., Methods: Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays., Results: Ibrolipim 5 and 50 μmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim., Conclusion: Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.