Liver X Receptors Agonist TO901317 Research Articles

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 h. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2 A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Liver X Receptor Agonist Inhibits Oxidized Low-Density Lipoprotein Induced Choroidal Neovascularization via the NF-κB Signaling Pathway

Age-related macular degeneration (AMD) is the most common blindness-causing disease among the elderly. Under oxidative stress, low-density lipoprotein in the outer layer of the retina is easily converted into oxidized low-density lipoprotein (OxLDL), which promotes the development of choroidal neovascularization (CNV), the main pathological change in wet AMD. Liver X receptor (LXR), a ligand-activated nuclear transcription factor, regulates various processes related to CNV, including lipid metabolism, cholesterol transport, inflammation, and angiogenesis. In this study, we evaluated the effects of the LXR agonist TO901317 (TO) on CNV. Our results demonstrated that the TO could inhibit OxLDL-induced CNV in mice as well as inflammation and angiogenesis in vitro. Using siRNA transfection in cells and Vldlr-/- mice, we further confirmed the inhibitory effects of TO against the inflammatory response and oxidative stress. Mechanistically, the LXR agonist reduces the inflammatory response via the nuclear translocation of NF-κB p65 in the pathway for NF-κB activation and by enhancing ABCG1-dependent lipid transportation. Therefore, an LXR agonist is a promising therapeutic candidate for AMD, especially for wet AMD.

Effect of liver X receptor agonist TO901317 on cognitive function in APP/PS1 double transgenic mice with Alzheimer's disease and the underlying mechanism.

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily, and LXR-β is an important receptor for cholesterol content in brain cells. LXR-β/retinoic X receptor (RXR-α)/ATP binding cassette transporter A1 (ABCA1) cholesterol transmembrane transport system is closely related to the occurrence and development of Alzheimer's disease (AD). LXR agonist TO901317 can affect the accumulation of β- amyloid protein in the brain tissue of APP/PS1 double transgenic AD mice. However, the molecular mechanism is not clarified in detail. This study aims to evaluate the effects of LXR agonist TO901317 on the cognitive function of AD mice fed with high cholesterol diet, and to explore its possible mechanism from the perspective of cholesterol metabolism. Twenty four male 6-month-old APP/PS1 double transgenic AD mice were randomly divided into 4 groups, 6 mice in each group: a control group (fed with normal diet), a cholesterol rich diet (CRD) group, a TO901317 group (fed with CRD combined with TO901317), and a GSK2033 group (fed with CRD combined with TO901317 and LXR antagonist GSK2033). The mice were fed with pellet feed made of high cholesterol feed, mixed with lard, egg yolk powder, and cod liver oil twice a day. TO901317 and GSK2033 were dissolved and diluted to a final concentration at 0.03%. The drugs were given to the mice daily through gastric tube according to their body weight. Meanwhile, the mice in the drug group were fed with high cholesterol diet . After feeding for 3 months, Morris water maze was used to observe the changes of spatial exploration and memory ability of AD mice in each group. The contents of TC, LDL, and HDL in serum of mice in each group were detected by cholesterol enzyme colorimetry, and the differences among the groups were compared. The expression of Aβ42 in the brain of AD mice was detected by ELISA. Western blotting was used to detect the protein levels of LXR-β, RXR-α, ABCA1, and Caveolin-1 in the brain of each group. Morris water maze results showed that the times, distance and the duration of mice crossing the platform in the CRD group were significantly decreased compared with the control group (all P<0.05), while these three figures in TO901317 group were significantly increased compared with the CRD group (all P<0.05). Compared with the TO901317 group, there was a decrease of these figures in the GSK2033 group (all P<0.05). The serum TC and LDL levels in the CRD group were significantly higher than those in the control group, while HDL levels were significantly lower (all P<0.001). The figures of the TC and LDL contents level in the TO901317 group were lower than those in the CRD group, while HDL levels were higher (all P<0.001). Compared with TO901317 group, the contents of the TC and LDL in GSK2033 group were significantly increased, while HDL content was significantly decreased (all P<0.001). ELISA results showed that the production of Aβ42 peptides in the brain of CRD group was the highest while the content in the TO901317 group was significantly decreased (P<0.001), which was the lowest among the groups. The figure in the control group was close to the GSK2033 group. Western blotting results showed that the protein levels of LXR-β, RXR-α, and ABCA1 in the CRD group were significantly decreased compared with the control group, but the protein level of Caveolin-1 was increased (all P<0.01). After TO901317 treatment, the protein levels of LXR-β, RXR-α and ABCA1 were significantly increased, while the protein level of Caveolin-1 was decreased partially (all P<0.001). In the GSK2033 group, the effect of TO901317 on AD mice was partially reversed by GSK2033. Compared to TO901317 group, the protein levels of LXR-β, RXR-α, and ABCA1 showed a decrease trend, while the protein level of Caveolin-1 showed an increase state (all P<0.05). High cholesterol diet leads to severer spatial exploration, learning and memory impairment in transgenic AD mice, while the LXR agonist TO901317 attenuates this effect. The mechanism may be that TO901317 promotes cholesterol efflux by activating LXR-β/RXR-α/ABCA1 transmembrane transport system, reduces the expression of Caveolin-1, improves the composition of lipid raft, and ultimately reduces the production of Aβ42 in the brain.

Liver X Receptor (LXR) Activation Decreases Chronic Myelogenous Leukemia Cell Viability and Alters the Expression of Antiapoptotic and Cholesterol Genes

Liver X receptors (LXRs) are oxysterol‐activated nuclear receptors involved in the regulation of cholesterol transport, glucose metabolism, and inflammatory responses. Recently, LXRs have been shown to possess anti‐cancer effects in a variety of cancer types, in part due its capacity to deplete essential lipids required for cell proliferation. However, the role of LXRs in chronic myelogenous leukemia (CML) has yet to be elucidated. In this study, we investigated the effects of LXR activation on the human erythroleukemia K562 cell line – a model for CML. K562 cells were treated with the LXR agonist TO901317 (0μM, 1μM, 5μM, 10μM) to determine dose‐ and time‐dependent effects on cell viability, cell number and size, and the mRNA expression of genes related to cholesterol metabolism (ATP‐binding cassette transporter A1 (ABCA1), low‐density lipoprotein receptor (LDLR), and HMG‐CoA reductase (HMGCR)) and anti‐apoptosis (B cell leukemia‐xL (Bcl‐xL)). TO901317 treatment significantly increased ABCA1 mRNA expression at 24hr and 72hr, with greater expression at the lowest treatment concentration (1μM). HMGCR and Bcl‐xL mRNA expression was dose‐dependently reduced by LXR activation after 72hr, as were decreases in total cell number. Average cell size similarly decreased from 24hr to 72hr with 10μM TO901317 treatment. Further, K562 viability was decreased by TO901317 treatment at all concentrations at 24hr, 48hr, and 72hr, with dose‐dependent effects observed at 48hr only. Together, these findings suggest that LXR agonism decreases CML cell viability and number, and that these changes may be related to modulation of cholesterol metabolism and apoptotic pathways. Current studies are underway to further investigate these pathways, in addition to the capacity of TO901317‐induced LXR activation to modulate cellular differentiation.Support or Funding InformationThe Science Institute of the College of Arts and Sciences and Faculty Research Committee, Fairfield University

Identification of a Novel Liver X Receptor Agonist that Regulates the Expression of Key Cholesterol Homeostasis Genes with Distinct Pharmacological Characteristics.

Activation of liver X receptor (LXR) is associated with cholesterol metabolism and anti-inflammatory processes, which makes it beneficial to antiatherosclerosis therapy. Nevertheless, existing agonists that target LXR, for example TO901317, are related to unwanted side effects. In the present study, using a screening method we identified IMB-808, which displayed potent dual LXRα/β agonistic activity. In vitro, IMB-808 effectively increased the expressing quantity of genes related to reverse cholesterol transport process as well as those associated with cholesterol metabolism pathway in multiple cell lines. Additionally, IMB-808 remarkably promoted cholesterol efflux from RAW264.7 as well as THP-1 macrophage cells and reduced cellular lipid accumulation accordingly. Interestingly, compared with TO901317, IMB-808 almost did not increase the expressing quantity of genes related to lipogenesis in HepG2 cells, which indicated that IMB-808 could exhibit fewer internal lipogenic side effects with a characteristic of selective LXR agonist. Furthermore, in comparison with the full LXR agonist TO901317, IMB-808 recruits coregulators differently and possesses a distinct predictive binding pattern for the LXR ligand-binding domain. In summary, our study demonstrated that IMB-808 could act as an innovative partial LXR agonist that avoids common lipogenic side effects, providing insight for the design of novel LXR modulators. Our data indicate that this compound might be used as a promising therapeutic agent for the prospective treatment of atherosclerosis in the future.

Liver X Receptor Agonist TO901317 Attenuates Paraquat-Induced Acute Lung Injury through Inhibition of NF-κB and JNK/p38 MAPK Signal Pathways.

Paraquat (PQ) is a widely used herbicide with extremely high poisoning mortality mostly from acute lung injury (ALI) or progressive pulmonary fibrosis. Toxicity mechanisms remain unclear, but a redox cycling process that generates reactive oxygen species (ROS) is involved, as are inflammation and cell apoptosis. We established an ALI mouse model by intraperitoneal injection of PQ (28 mg/kg) and then investigated the effects of a potent liver X receptor (LXR) agonist, TO901317 (5 or 20 mg/kg), injected intraperitoneally 30 min after PQ administration. Poisoned mice exhibited severe lung tissue lesions and edema, significant neutrophilic (PMNs) infiltration, and release of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). PQ administration also decreased activity of antioxidases, including superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferases (GSTs), and increased lipid peroxidation as evaluated by malondialdehyde (MDA) levels. PQ exposure induced upregulation of the proapoptotic gene Bax and downregulation of the antiapoptotic gene Bcl-2, leading to marked cell apoptosis in the lung tissues. TO901317 treatment reversed all these effects through inhibition of PQ-induced nuclear factor kappa B (NF-κB) and JNK/p38 mitogen-activated protein kinase (MAPK) activation. The LXR agonist TO901317 had potent antioxidant, anti-inflammatory, and antiapoptotic effects against PQ-induced ALI.

LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone

Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the external granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects.

Soluble (pro)renin receptor via β-catenin enhances urine concentration capability as a target of liver X receptor

The extracellular domain of the (pro)renin receptor (PRR) is cleaved to produce a soluble (pro)renin receptor (sPRR) that is detected in biological fluid and elevated under certain pathological conditions. The present study was performed to define the antidiuretic action of sPRR and its potential interaction with liver X receptors (LXRs), which are known regulators of urine-concentrating capability. Water deprivation consistently elevated urinary sPRR excretion in mice and humans. A template-based algorithm for protein-protein interaction predicted the interaction between sPRR and frizzled-8 (FZD8), which subsequently was confirmed by coimmunoprecipitation. A recombinant histidine-tagged sPRR (sPRR-His) in the nanomolar range induced a remarkable increase in the abundance of renal aquaporin 2 (AQP2) protein in primary rat inner medullary collecting duct cells. The AQP2 up-regulation relied on sequential activation of FZD8-dependent β-catenin signaling and cAMP-PKA pathways. Inhibition of FZD8 or tankyrase in rats induced polyuria, polydipsia, and hyperosmotic urine. Administration of sPRR-His alleviated the symptoms of diabetes insipidus induced in mice by vasopressin 2 receptor antagonism. Administration of the LXR agonist TO901317 to C57/BL6 mice induced polyuria and suppressed renal AQP2 expression associated with reduced renal PRR expression and urinary sPRR excretion. Administration of sPRR-His reversed most of the effects of TO901317. In cultured collecting duct cells, TO901317 suppressed PRR protein expression, sPRR release, and PRR transcriptional activity. Overall we demonstrate, for the first time to our knowledge, that sPRR exerts antidiuretic action via FZD8-dependent stimulation of AQP2 expression and that inhibition of this pathway contributes to the pathogenesis of diabetes insipidus induced by LXR agonism.

Pharmacological Activation of LXRs Decreases Amyloid-&amp;#946; Levels in Niemann-Pick Type C Model Cells

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPβ levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid β (Aβ) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aβ levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aβ levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aβ levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

Hormonal modulators of glial ABCA1 and apoE levels

Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.

Liver X Receptor Agonist Alleviated High Glucose‐Induced Endothelial Progenitor Cell Dysfunction via Inhibition of Reactive Oxygen Species and Activation of AMP‐Activated Protein Kinase

Liver X receptors (LXRs) are key regulators of cholesterol homeostasis. Synthetic LXR agonists are anti-atherogenic and anti-inflammatory. However, the effect of LXR agonists on endothelial progenitor cell (EPC) function is largely unknown. Here, we explored the effect of the LXR agonist TO901317 (TO) on EPC biology and the underlying mechanisms. Endothelial progenitor cells were cultured in mannitol or 30 mm glucose (high glucose) for 24 hours. For TO treatments, cells were pretreated with TO (10 μm) for 12 hours, then mannitol or high glucose was added for an additional 24 hours. EPCs function, reactive oxygen species (ROS) release, and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) were analyzed. TO could restore the high glucose-impaired adhesion and migration capacity of EPCs. High glucose impaired EPC-mediated angiogenesis, and TO reversed the impairment. TO also alleviated ROS release induced by high glucose. Western blot analysis revealed that high glucose downregulated the phosphorylation of AMPK and endothelial nitric oxide synthase, which could be reversed with TO treatment. Furthermore, inhibiting AMPK activation by compound C could abolish the protective effects of TO on EPCs. TO had a protective effect on EPCs under high glucose by inhibiting ROS release and activating AMPK.

Abstract 3494: Liver X receptor activation represents a novel strategy to block Hedgehog signaling in pancreatic adenocarcinoma and enhance the antitumor effect of gemcitabine

Abstract Background: The Hedgehog (Hh) signaling pathway plays a pathogenic role in many human cancers, including pancreatic adenocarcinoma, and several pathway antagonists have begun clinical testing. All of these novel agents target Smoothened (Smo), a key regulator of Hh signaling, but reported emergence of resistance mutations questions the durability of this approach. Oxysterols, oxidized derivatives of cholesterol, can modulate Hh signaling either through direct interaction with Smo or via cross-talk with liver X receptors (LXR) that serve as their natural receptors. Thus, oxysterols may represent mechanistically novel Hh inhibitors, especially if they involve LXRs. Methods: We examined the effects of LXR activation on the in vivo growth of pancreatic cancer. Nude mice bearing pancreatic cancer xenografts derived from primary clinical specimens were treated with a vehicle control, gemcitabine, the non-steroidal LXR agonist TO901317, or the combination of gemcitabine and TO901317 for 4-6 weeks. The LXR agonist TO901317 was used since it lacks the ability to directly bind to Smo. Tumor volumes were measured to assess response, and LXR and Hh pathway activities were quantified by real-time RT-PCR for Hh (GLI1, PTCH1) and LXR (ABCA1) target gene expression. Human specific primers were used to detect changes in tumor cells, whereas mouse-specific primers were used to study stroma. Intratumoral levels of gemcitabine triphosphate were quantified by LC-MS-MS. Results: Administration of TO901317 alone did not impact tumor growth compared to control treated animals similar to previous findings with Smo antagonists. Treatment with gemcitabine alone decreased tumor growth, However the addition of TO901317 to gemcitabine significantly enhanced this effect to promote tumor regression. In vivo treatment with TO901317 successfully activated LXR in both human tumor cells and murine stromal cells as evidenced by increased expression of ABCA1. Moreover, TO901317 inhibited expression of the Hh targets GLI1 and PTCH1 5- and 2-fold, respectively, in stromal cells and 5-fold in tumor cells. Previous studies (Olive et al, Science, 2009) demonstrated that Hh pathway inhibition improves drug delivery in vivo. Therefore, we quantified intratumoral levels of gemcitabine triphosphate, the active metabolite of gemcitabine, and found that it was significantly elevated in tumors exposed to TO901317 (P&amp;lt;0.03). Conclusions: The LXR agonist TO901317 enhances the cytotoxic effects of gemcitabine to promote tumor regression in a human xenograft model of pancreatic adenocarcinoma. Therefore, LXR agonists, such as TO901317 and oxysterols, may represent a novel strategy to target pathologic Hh signaling within both tumor cells as well as the surrounding stroma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3494. doi:1538-7445.AM2012-3494

Modified Low Density Lipoprotein Stimulates Complement C3 Expression and Secretion via Liver X Receptor and Toll-like Receptor 4 Activation in Human Macrophages

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRβ in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.

TO901317 regulating apolipoprotein M expression mediates via the farnesoid X receptor pathway in Caco-2 cells

BackgroundApolipoprotein M (apoM) may have potential antiatherosclerotic properties. It has been reported that apoM expression could be regulated by many intracellar and extracellar factors. In the present study we further investigated regulation of apoM expression in Caco-2 cells stimulated by a liver X receptor (LXR) agonist, TO901317.Materials and methodsCaco-2 cells were cultured in the presence of either TO901317, farnesoid X receptor (FXR) antagonist guggulsterone or TO901317 together with guggulsterone at different concentrations for 24 hrs. The mRNA levels of ATP-binding cassette transporter A1 (ABCA1), apoA1, apoM, liver receptor homologue-1 (LRH-1) and short heterodimer partner 1 (SHP1) were determined by real-time RT-PCR.ResultsWhen Caco-2 cell cultured with TO901317 alone, the mRNA levels of ABCA1, apoA1, apoM, LRH-1 and SHP1 were significantly increased with dose-dependent manners (p < 0.05), whereas when the cells cultured with guggulsterone alone, the mRNA levels of apoM, SHP1 and LRH-1 (p < 0.05) were strongly inhibited. Moreover, guggulsterone could abolish the TO901317 enhanced mRNA levels of apoA1 apoM, SHP1 and LRH-1.ConclusionThe present study demonstrated that LXR agonist TO901317 induced apoM expression in Caco-2 cells might be mediated via the LXR/FXR pathway.

Critical Role of Astroglial Apolipoprotein E and Liver X Receptor-α Expression for Microglial Aβ Phagocytosis

Liver X receptors (LXRs) regulate immune cell function and cholesterol metabolism, both factors that are critically involved in Alzheimer's disease (AD). To investigate the therapeutic potential of long-term LXR activation in amyloid-β (Aβ) peptide deposition in an AD model, 13-month-old, amyloid plaque-bearing APP23 mice were treated with the LXR agonist TO901317. Postmortem analysis demonstrated that TO901317 efficiently crossed the blood-brain barrier. Insoluble and soluble Aβ levels in the treated APP23 mice were reduced by 80% and 40%, respectively, compared with untreated animals. Amyloid precursor protein (APP) processing, however, was hardly changed by the compound, suggesting that the observed effects were instead mediated by Aβ disposal. Despite the profound effect on Aβ levels, spatial learning in the Morris water maze was only slightly improved by the treatment. ABCA1 (ATP-binding cassette transporter 1) and apolipoprotein E (ApoE) protein levels were increased and found to be primarily localized in astrocytes. Experiments using primary microglia demonstrated that medium derived from primary astrocytes exposed to TO901317 stimulated phagocytosis of fibrillar Aβ. Conditioned medium from TO901317-treated ApoE(-/-) or LXRα(-/-) astrocytes did not increase phagocytosis of Aβ. In APP23 mice, long-term treatment with TO901317 strongly increased the association of microglia and Aβ plaques. Short-term treatment of APP/PS1 mice with TO901317 also increased this association, which was dependent on the presence of LXRα and was accompanied by increased ApoE lipidation. Together, these data suggest that astrocytic LXRα activation and subsequent release of ApoE by astrocytes is critical for the ability of microglia to remove fibrillar Aβ in response to treatment with TO901317.

Liver X receptor agonist inhibits proliferation of ovarian carcinoma cells stimulated by oxidized low density lipoprotein

Objectives We previously observed an association between ovarian cancer outcome and statin use and hypothesized lipoproteins have direct effects on ovarian cancer proliferation. Here we investigate the direct effects of low density lipoprotein (LDL) and oxidized LDL (oxLDL) on proliferation and the inhibitory effects of fluvastatin and a liver X receptor (LXR) agonist. Methods The effects of LDL, oxLDL, the LXR agonist TO901317, fluvastatin and cisplatin on cellular proliferation were determined using MTT assays. LXR pathway proteins were assayed by immunoblotting. Cytokine expression was determined by antibody array. Results Concentrations of oxLDL as small as 0.1 μg/ml stimulated CAOV3 and SKOV3 proliferation, while LDL had no effect. TO901317 inhibited the proliferation of CAOV3, OVCAR3 and SKOV3 cells stimulated by oxLDL. Fluvastatin inhibited oxLDL mediated proliferation of CAOV3 and SKOV3. Cardiotrophin 1 (CT-1) was mitogenic to CAOV3 and SKOV3, was induced by oxLDL, and was reversed by TO901317. OxLDL increased cisplatin IC50s by 3.8 μM and > 60 μM for CAOV3 and SKOV3 cells, respectively. The LXR pathway proteins CD36, LXR, and ABCA1 were expressed in eight ovarian carcinoma cell lines (A2780, CAOV3, CP70, CSOC882, ES2, OVCAR3, SKOV3). Conclusions OxLDL reduced ovarian carcinoma cell chemosensitivity and stimulated proliferation. These effects were reversed by LXR agonist or fluvastatin. The LXR agonist also inhibited expression of the ovarian cancer mitogen CT-1. These observations suggest a biologic mechanism for our clinical finding that ovarian cancer survival is associated with statin use. Targeting LXR and statin use may have a therapeutic role in ovarian cancer.

OSBP Negatively Regulates ABCA1 Protein Stability

Oxysterol binding to liver X receptors (LXR) increases the transcription of genes involved in cholesterol efflux and disposal, such as ABCA1 (ATP-binding cassette transporter A1). Other cytoplasmic sterol-binding proteins could interact with this pathway by sequestering or delivering substrates and ligands. One potential regulator is OSBP (oxysterol-binding protein), which is implicated in the integration of sterol sensing/transport with sphingomyelin synthesis and cell signaling. Since these activities could impact the cholesterol efflux pathway, we examined whether OSBP was involved in LXR regulation and in expression and activity of ABCA1. Suppression of OSBP in Chinese hamster ovary cells by RNA interference resulted in increased ABCA1 protein expression and cholesterol efflux activity following induction with oxysterols or the synthetic LXR agonist TO901317. OSBP knockdown in J774 macrophages also increased ABCA1 expression in the presence and absence of LXR agonists. OSBP depletion did not affect ABCA1 mRNA levels or LXR activity. Rather, OSBP silencing increased the half-life of ABCA1 protein by 3-fold. Sphingomyelin synthesis was suppressed in OSBP-depleted cells treated with 25-hydroxycholesterol but not TO901317 or 22-hydroxycholesterol and did not correlate with ABCA1 stabilization. Moreover, co-transfection experiments revealed that reduction of ABCA1 protein by OSBP was prevented by a mutation in the sterol-binding domain but not by mutations that abrogated interaction with the Golgi apparatus or endoplasmic reticulum. Thus, OSBP opposes the activity of LXR by negatively regulating ABCA1 activity in the cytoplasm by sterol-binding domain-dependent protein destabilization.

LXRα Enhances Lipid Synthesis in SZ95 Sebocytes

Differentiation of sebocytes is strongly associated with enhanced lipid synthesis and accumulation in the cells. Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which play a critical role in cholesterol homeostasis and lipid metabolism. We examined whether LXRalpha regulated lipid synthesis in the immortalized human sebaceous gland cell line SZ95. When the SZ95 sebocytes were treated with the ligand of LXR such as TO901317 or 22(R)-hydroxycholesterol, lipid droplets were accumulated in the majority of cells when examined by Oil Red O staining. The expression of the known LXR targets, such as fatty acid synthase and sterol regulatory-binding protein-1, was induced by TO901317. TO901317 treatment increased expression of LXRalpha but not that of LXRbeta. Transfection of antisense LXRalpha significantly decreased TO901317-induced target gene expression and lipid droplet accumulation, suggesting a major role of LXRalpha in differentiation of sebocytes. Further, TO901317 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase that was induced by lipopolysaccharide treatment. Together, these results indicate that important roles of LXRalpha in differentiation and inflammatory signaling in sebaceous glands. Thus, we suggest that LXR ligands could provide a new class of therapeutic agents for sebaceous gland-associated disorders such as seborrhea and acne.

Dual Mechanisms for the Fibrate-mediated Repression of Proprotein Convertase Subtilisin/Kexin Type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.

Regulation of central nervous system cholesterol homeostasis by the liver X receptor agonist TO-901317

The nuclear oxysterol receptors known as liver X receptors (LXR) are responsible for regulating genes involved in cholesterol homeostasis. Two subtypes exist, LXR-alpha and LXR-beta, both of which are expressed in the central nervous system (CNS). Activation of LXR causes an increase in mechanisms involved in cholesterol efflux, including ATP-binding cassette transporters (ABC)-A1 and ABC-G1. Although LXR agonists have been found to induce gene expression for ABC-A1 and ABC-G1 in the CNS, no functional response has been recorded. In this study, we show that an LXR agonist (TO-901317) increases protein levels of the cholesterol transporters ABC-A1 and ABC-G1, as well as the cholesterol chaperone protein apolipoprotein E (apoE). These changes are associated with a decrease in cholesterol levels from isolated cortical synaptosomal plasma membranes. LXR activation occurs in response to increased cholesterol levels in cells. However, while data exists on how LXR alter cholesterol efflux, there is no data on whether these receptors alter production of cholesterol. We found that TO-901317 increases HMG-CoA reductase activity, the rate-limiting step of cholesterol production in vivo. This finding was duplicated in an in vitro system. Although high concentrations (>1 microM) of TO-901317 were required to induce HMG-CoA reductase activity, these changes only occurred in the presence of apolipoprotein AI, suggesting that cholesterol efflux is required for this effect to occur. These data show that the LXR agonist TO-901317 is capable of reducing cholesterol in neurons of the CNS.