LXR Agonist Research Articles

Regulation of liver X receptor target genes by 22-functionalized oxysterols. Synthesis, in silico and in vitro evaluations

The endogenous oxysterol 22(R)-hydroxycholesterol (22RHC, 1) is an LXR agonist which upregulates genes of critical involvement in human cholesterol- and lipid metabolism. In contrast, its synthetic epimer 22(S)-hydroxycholesterol (22SHC, 8) has shown specific antagonistic effects in recent studies, avoiding unwanted side effects provided by potent LXR agonists. In terms of LXR modulation, the aim of this study was to compare 22SHC (8), 22RHC (1) and synthesized ligands with keto- and amide functionality in the 22nd position of the cholesterol scaffold. 22SHC (8) and 22RHC (1) performed as expected while 22-ketocholesterol (22KC, 10) revealed an attractive in vitro profile for further investigation in terms of anti-atherosclerotic properties as selective upregulation of the ATP-binding cassette transporter ABCA1 was observed. A new synthesized amide derivate, Fernholtz cyclohexylamide (13) was shown to reduce lipogenesis in a dose-responsive manner and abolish the effect of the potent LXR agonist T0901317 when administered simultaneously.

Luteolin improves non-alcoholic fatty liver disease in db/db mice by inhibition of liver X receptor activation to down-regulate expression of sterol regulatory element binding protein 1c

In this study, we report that daily administration of luteolin for 8 weeks improved hepatic steatosis by repressing hepatic TG accumulation and increasing glycogen storage. Luteolin inhibited hepatic de novo lipid synthesis by regulating the LXR-SREBP-1c signaling pathway, which is over-activated in the livers of db/db mice. Further in vitro studies revealed that luteolin can competitively bind to the ligand binding domain to suppress the LXR activation induced by an LXR agonist and high glucose, thereby decreasing TG accumulation in HepG2 cells and primary hepatocytes. Taken together, our results indicate that luteolin can abolish lipid accumulation induced by LXR-SREBP-1c activation both in vivo and in vitro, and may have potential as a therapeutic agent for treating NAFLD.

The effect of a liver-X-receptor ligand on bleomycin induced pulmonary fibrosis in mice

The liver-X-receptors have shown anti-fibrosis ability in several animal models. Our purpose was to investigate the effect of LXRs in bleomycin induced lung fibrosis in mice. Bleomycin was intratracheally delivered to mice. Some mice were administered a LXR agonist, T0901317. Then mice were evaluated for the development of lung inflammation and fibrosis. T0901317 was able to attenuate the inflammation and fibrosis induced by bleomycin. T0901317 treatment evidently abolished the high level of TGF-β1 and inhibited NF-κB DNA-binding activity in lung. So LXRs may attenuate the progressing of lung fibrosis, providing a potential treatment of IPF.

Liver X Receptors and their Agonists: Targeting for Cholesterol Homeostasis and Cardiovascular Diseases.

Liver X receptors α (LXRα) and β (LXRβ) are essential for protection against cardiovascular diseases. LXRs are members of the nuclear receptor superfamily of DNA-binding transcription factors and act as sensors of cholesterol homeostasis. In this review, we introduce LXRs and briefly describe the roles of LXRs in reverse cholesterol transport and trans-intestinal cholesterol efflux. We discuss LXR agonists and the downstream genes of LXRs that are involved in the regulation of cholesterol transport. In addition, we describe the cardioprotective effects of LXRs against atherosclerosis, myocardial ischemia/reperfusion injury, diabetic cardiomyopathy, and myocardial hypertrophy. Finally, we expand our discussion to the actions of LXRs in atherosclerosis and suggest several potential research avenues that may be of interest to clinicians and basic scientists. The information included herein may be useful for the design of future experimental research studies and may advance the investigation of LXRs as therapeutic targets.

Beneficial and Adverse Effects of an LXR Agonist on Human Lipid and Lipoprotein Metabolism and Circulating Neutrophils

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.

Cloning and Characterization of Lxr and Srebp1, and Their Potential Roles in Regulation of LC-PUFA Biosynthesis in Rabbitfish Siganus canaliculatus.

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability to biosynthesize C20-22 long-chain polyunsaturated fatty acid (LC-PUFA) from C18 PUFA precursors, which is generally absent or low in marine teleosts. Thus, understanding the molecular basis of LC-PUFA biosynthesis in rabbitfish will contribute to efforts aimed at optimizing LC-PUFA biosynthesis in teleosts, especially marine species. In the present study, the importance of the transcription factors liver X receptor (Lxr) and sterol regulatory element-binding protein 1 (Srebp1) in regulation of LC-PUFA biosynthesis in rabbitfish was investigated. First, full-length cDNA of Lxr and Srebp1 were cloned and characterized. The Lxr mRNA displayed a ubiquitous tissue expression pattern while Srebp1 was highly expressed in eyes, brain and intestine. In rabbitfish primary hepatocytes treated with Lxr agonist T0901317, the expression of Lxr and Srebp1 was activated, accompanied by elevated mRNA levels of Δ4 and Δ6/Δ5 fatty acyl desaturase (Fad), key enzymes of LC-PUFA biosynthesis, as well as peroxisome proliferator-activated receptor γ (PPARγ). In addition, Srebp1 displayed higher expression levels in liver of rabbitfish fed a vegetable oil diet or reared at 10 ppt salinity, which were conditions reported to increase the liver expression of Δ4 and Δ6/Δ5 Fad and LC-PUFA biosynthetic ability, than fish fed a fish oil diet or reared at 32 ppt, respectively. These results suggested that Lxr and Srebp1 are involved in regulation of LC-PUFA biosynthesis probably by promoting the expression of two Fad in rabbitfish liver, which, to our knowledge, is the first report in marine teleosts.

Obesity-related glomerulopathy: clinical and pathologic characteristics and pathogenesis.

The prevalence of obesity-related glomerulopathy is increasing in parallel with the worldwide obesity epidemic. Glomerular hypertrophy and adaptive focal segmental glomerulosclerosis define the condition pathologically. The glomerulus enlarges in response to obesity-induced increases in glomerular filtration rate, renal plasma flow, filtration fraction and tubular sodium reabsorption. Normal insulin/phosphatidylinositol 3-kinase/Akt and mTOR signalling are critical for podocyte hypertrophy and adaptation. Adipokines and ectopic lipid accumulation in the kidney promote insulin resistance of podocytes and maladaptive responses to cope with the mechanical forces of renal hyperfiltration. Although most patients have stable or slowly progressive proteinuria, up to one-third develop progressive renal failure and end-stage renal disease. Renin-angiotensin-aldosterone blockade is effective in the short-term but weight loss by hypocaloric diet or bariatric surgery has induced more consistent and dramatic antiproteinuric effects and reversal of hyperfiltration. Altered fatty acid and cholesterol metabolism are increasingly recognized as key mediators of renal lipid accumulation, inflammation, oxidative stress and fibrosis. Newer therapies directed to lipid metabolism, including SREBP antagonists, PPARα agonists, FXR and TGR5 agonists, and LXR agonists, hold therapeutic promise.

Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway

BackgroundLiver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. MethodsProstate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. ResultsData showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. ConclusionsThis study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway.

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Potential conflict of interest: Nothing to report. Reply: We would like to thank you for your interest in our article. We agree that our article, and the work of others,1 has shed a new light on the role of nuclear receptor signaling, and especially liver X receptor/retinoid X receptor (LXR/RXR) activation as a relatively early response to copper misbalance. We propose that drug‐mediated improvement of liver disease in the Wilson disease (WD) mice involves activation of LXR/RXR function not only in hepatocytes, but also in nonparenchymal liver cells (NPCs). Our findings suggest that copper overload negatively regulates LXR and RXR. The mechanisms underlying this regulation by copper remain to be fully elucidated. However, the implication that NPCs are involved in the pathogenesis of WD is an exciting new finding. Investigation of the cross‐talk between hepatocytes and stellate cells/Kupffer cells in this animal model may have ramifications for all fibrotic liver diseases. Although T0901317 was first reported to be a specific LXR agonist,4 we agree that the drug may target other nuclear receptors that form heterodimers with RXR. In fact, it could be that the pleotropic effect of the drug accounts for beneficial anti‐inflammatory and antifibrotic effects that we observe in WD mice. Identifying all targets of T0901317 in the WD model is an important future goal. Future modifications of the drug and/or careful evaluation of dosage/time of treatment are also likely to be necessary to offset the potential untoward effects (such as hepatic steatosis). Nonetheless, modulation of LXR activity appears to have promise as a novel therapy for WD.

Liver X Receptor Agonist TO901317 Prevents Diacylglycerols Accumulation in the Heart of Streptozotocin-Diabetic Rats

Background/Aims: Liver X receptors (LXRα and LXRβ) are ligand-activated transcription factors that regulate expression of genes involved in lipid and cholesterol metabolism. LXR expression has been identified in the heart, and enhanced LXR activity in the streptozotocin (STZ) diabetic myocardium was reported recently. The aim of this study was to investigate effect of in vivo LXR activation on myocardial lipid metabolism under conditions of STZ-induced diabetes. Methods: Wistar rats were randomly divided into three experimental groups: non-diabetic control, treated with STZ, and treated with STZ and LXR agonist - TO901317. Diabetes was induced by a single intraperitonal injection of STZ at a dose of 55 mg/kg. LXR agonist was administrated once daily in the morning by an oral gavage at a dose of 10 mg/kg/d during the last week of the experiment. After anesthesia samples of blood and the left ventricle were taken. Results: TO901317 administration increased expression of both LXR isoforms and its target genes: sterol response element binding protein 1c (SREBP-1c) and acetyl-coenzyme A carboxylase 1 (ACC1) in the heart of streptozotocin-diabetic rats. Treatment with LXR agonist had no effect on plasma lipids and glucose in the diabetic rats. Concomitantly, content of the examined lipid classes in the diabetic heart (nonesterified fatty acids, triacylglycerols, phospholipids, cholesterol esters, ceramide) was unchanged after treatment with TO901317. On the contrary, myocardial level of cholesterol and diacylglycerols (DAG) was decreased after LXR activation in diabetic rats, the change in DAG level was associated with downregulated expression of adipose triglyceride lipase (ATGL). Conclusion: Activation of LXRs by TO901317 protects cardiomyocytes against DAG accumulation and thus may reverse disturbances in lipid metabolism observed in streptozotocin-diabetic heart.

Role of Liver X Receptor in AD Pathophysiology.

Alzheimer's disease (AD) is the major cause of dementia worldwide. The pharmacological activation of nuclear receptors (Liver X receptors: LXRs or Retinoid X receptors: RXR) has been shown to induce overexpression of the ATP-Binding Cassette A1 (ABCA1) and Apolipoprotein E (ApoE), changes that are associated with improvement in cognition and reduction of amyloid beta pathology in amyloidogenic AD mouse models (i.e. APP, PS1: 2tg-AD). Here we investigated whether treatment with a specific LXR agonist has a measurable impact on the cognitive impairment in an amyloid and Tau AD mouse model (3xTg-AD: 12-months-old; three months treatment). The data suggests that the LXR agonist GW3965 is associated with increased expression of ApoE and ABCA1 in the hippocampus and cerebral cortex without a detectable reduction of the amyloid load. We also report that most cells overexpressing ApoE (86±12%) are neurons localized in the granular cell layer of the hippocampus and entorhinal cortex. In the GW3965 treated 3xTg-AD mice we also observed reduction in astrogliosis and increased number of stem and proliferating cells in the subgranular zone of the dentate gyrus. Additionally, we show that GW3965 rescued hippocampus long term synaptic plasticity, which had been disrupted by oligomeric amyloid beta peptides. The effect of GW3965 on synaptic function was protein synthesis dependent. Our findings identify alternative functional/molecular mechanisms by which LXR agonists may exert their potential benefits as a therapeutic strategy against AD.

Liver X Receptor Regulates Triglyceride Absorption Through Intestinal Down-regulation of Scavenger Receptor Class B, Type 1

Reducing postprandial triglyceridemia may be a promising strategy to lower the risk of cardiovascular disorders associated with obesity and type 2 diabetes. In enterocytes, scavenger receptor class B, type 1 (SR-B1, encoded by SCARB1) mediates lipid-micelle sensing to promote assembly and secretion of chylomicrons. The nuclear receptor subfamily 1, group H, members 2 and 3 (also known as liver X receptors [LXRs]) regulate genes involved in cholesterol and fatty acid metabolism. We aimed to determine whether intestinal LXRs regulate triglyceride absorption. C57BL/6J mice were either fed a cholesterol-enriched diet or given synthetic LXR agonists (GW3965 or T0901317). We measured the production of chylomicrons and localized SR-B1 by immunohistochemistry. Mechanisms of postprandial triglyceridemia and SR-B1 regulation were studied in Caco-2/TC7 cells incubated with LXR agonists. In mice and in the Caco-2/TC7 cell line, LXR agonists caused localization of intestinal SR-B1 from apical membranes to intracellular organelles and reduced chylomicron secretion. In Caco-2/TC7 cells, LXR agonists reduced SR-B1-dependent lipidic-micelle-induced Erk phosphorylation. LXR agonists also reduced intracellular trafficking of the apical apolipoprotein B pool toward secretory compartments. LXR reduced levels of SR-B1 in Caco-2/TC7 cells via a post-transcriptional mechanism that involves microRNAs. In Caco-2/TC7 cells and mice, intestinal activation of LXR reduces the production of chylomicrons by a mechanism dependent on the apical localization of SR-B1.

Abstract 9805: Lack of Correlation Between HDL Level and Fecal Cholesterol in Multiple Mouse Models Suggests Importance of HDL Functionality in Atherosclerosis Attenuation

To circumvent the residual cardiovascular disease burden in patients receiving statin therapy, HDL-raising was suggested as a plausible approach. However, lack of benefits of CETP inhibitors, Torcetrapib and Dalcetrapib, despite substantial increase in HDL, and lack of efficacy of CER-001, an apoA-I mimetic, proved disappointing. Paradoxically, apoA-I Milano subjects with low HDL have reduced CVD risk. These findings shifted the focus from HDL level to HDL function. Promoting reverse cholesterol transport (RCT) is one of the main functions of HDL, which is measured in an in vivo assay as macrophage-derived fecal 3H-cholesterol. Non-radioisotopic method to quantitate fecal cholesterol (chol) as an RCT index was found to correlate with macrophage radioisotopic method in mice. Using this method, we carried out a number of studies in several mouse models, including C57Bl, apoA-I-Tg, LDLr KO, and ob/ob, using tool compounds, LXR and PPAR-α agonists, known to enhance RCT. Animals were treated with LXR and PPAR-α agonists alone or in combination, and lipid profile, HDL level, serum chol efflux, fecal chol, and, in appropriate animal models, aortic lipid contents were measured. In C57Bl mice, LXR agonist, T1317, raised HDL by 30%, while feno decreased HDL by 30%, but fecal chol showed a 2-fold increase in both cases. Interestingly, combination (comb) treatment with LXR and PPAR-α agonists did not change HDL, but fecal chol increased by 4.5-fold. HDL in apoA-I-Tg mice increased by 40%, 80%, and 80% in LXR, PPAR-α, and comb treated groups; respective fecal chol increases were 1.8, 1.8, and 5-fold, suggesting a lack of correlation between HDL level and fecal chol. LXR, PPAR-α, and comb-treated ob/ob mice showed 40%, 35%, and 40% rise in HDL, but 3-, 3-, and 20-fold increases in fecal chol, respectively. No correlations in HDL level and fecal chol contents were noticed in LDLr KO mice as well. Atherosclerosis attenuation by LXR and PPAR-α agonists in LDLr KO and ob.ob/LDLr DKO mice was associated with increased fecal chol, but not HDL level. These data suggest: a) a lack of correlation between HDL level and fecal chol; b) synergistic effect of LXR and PPAR-α agonists on RCT; c) reduced lesion area associated with increased fecal chol; and d) RCT influenced by additional mechanisms.

Abstract 13712: Serum HDL Cholesterol Decreases in Microrna-33b Knock-in Mice for an Intron of Sterol Regulatory Element-binding Factor 1 (Srebf1)

Background: MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33 (miR-33a) located within the intron of sterol regulatory element-binding factor (SREBF) 2 controls cholesterol homeostasis. Primates, but not rodents, express a second miR-33 gene (miR-33b) from an intron of SREBF1. To address miR-33b function in vivo, we developed humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron. Methods: The human miR-33b sequence was introduced into intron 16 of mouse Srebf1 because miR-33b is located in intron 16 of human SREBF1 and there are high homologies in exons 16 and 17 between human and mouse. The expression of serum miRNA was normalized with cel-miR-39 as a spike-in control. Results: We successfully established miR-33b knock-in (KI) mice with C57BL/6 background and this miR-33b KI strategy did not alter Srebf1 intron 16 splicing, which was confirmed by RT-PCR and sequencing. An LXR agonist T0901317, which induces Srebf1 expression, enhanced miR-33b expression in primary hepatocytes and the liver of miR-33 KI mice. The protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. Peritoneal macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Serum HDL-C levels were reduced by almost 35% in miR-33b KI mice. HPLC elution analysis showed that the decreased HDL-C levels were mainly composed of very large-, large-, medium sized HDL-C, which was compatible with the previous results of miR-33a deficient mice. Next, we measured miR-33b levels in serum of human. The expressions of miR-33b-3p were inversely correlated with serum HDL-C levels (P=0.025, R=0.336). Conclusions: miR-33b KI mice for an intron of Srebf1 showed reduced HDL-C level and serum miR-33b-3p levels were inversely correlated with HDL-C levels in human. These results indicate that miR-33b can be a potential target for raising HDL-C and may account for lower HDL-C levels in humans than those in mice. These mice will aid in elucidating the roles of miR-33s and screening of the drugs that can alter miR-33s levels and activities.

Abstract 16204: Knock-down of 24-dehydrocholesterol Reductase Suppresses SREBP2: A Novel Approach to Reduce PCSK9 in vivo

Introduction: Current therapeutic approaches to suppress the actions of proprotein convertase subtilisin/kexin type 9 (PCSK9) primarily involve monoclonal antibody. An alternative approach could be to suppress the SREBP2 pathway. Inhibition of sterol regulatory element-binding protein cleacage-activating protein (SCAP) will prevent SREBP mediated transcription of sterol biogenesis genes and PCSK9.24-dehydrocholesterol reductase (DHCR24) catalyzes the last step of cholesterol synthesis, converting desmosterol to cholesterol. Desmosterol is a strong LXR agonist and an inhibitor of SCAP. Hypothesis: We tested our hypothesis that reduction of DHCR24 expression would result in an increase in desmosterol, resulting in the suppression of SCAP and reduction of PCSK9 in vivo. Methods: In these studies, we used AAVshRNA to knock-down DHCR24 in the livers of CETP transgenic mice and WT mice. Results: Animals injected with AAVshRNA-DHCR24 resulted in 85% knockdown of DHCR24, with a concomitant 75% suppression of cholesterol synthesis, as measured by deuterium incorporation into newly made cholesterol. Knock-down of DHCR24 was also associated with up-regulation of fatty acid oxidation genes and suppression of fatty acid biosynthetic and SREBP-2 cholesterol biosynthetic pathways. Interestingly, DHCR24 knockdown was associated with a 60% decrease in plasma PCSK9 levels in CETP Tg mice (n=10, P<0.001) and similar results were observed in WT mice. Conclusions: To our knowledge, this is the first in vivo study to demonstrate that PCSK9 levels can be decreased via suppression of DHCR24 in vivo . These results suggest that DHCR24 might represent a novel therapeutic target for reducing LDL-cholesterol.

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA-I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux.

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1-syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from Abca1(+/+) and Abca1(-/-) mice and show their phenotype recapitulates primary macrophages. Abca1(+/+) lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized Abca1(-/-) macrophages show no efflux to apoA-I. In response to LPS, Abca1(-/-) macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11-26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1-syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1-syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I.

Destabilization of the torsioned conformation of a ligand side chain inverts the LXRβ activity

BackgroundLiver X receptors (LXRs) are transcription factors activated by cholesterol metabolites containing an oxidized side chain. Due to their ability to regulate lipid metabolism and cholesterol transport, they have become attractive pharmacological targets. LXRs are closely related to DAF-12, a nuclear receptor involved in nematode lifespan and regulated by the binding of C-27 steroidal acids. Based on our recent finding that the lack of the C-25 methyl group does not abolish their DAF-12 activity, we evaluated the effect of removing it from the (25R)-cholestenoic acid, a LXR agonist. MethodsThe binding mode and the molecular basis of action of 27-nor-5-cholestenoic acid were evaluated using molecular dynamics simulations. The biological activity was investigated using reporter gene expression assays and determining the expression levels of endogenous target genes. The in vitro MARCoNI assay was used to analyze the interaction with cofactors. Results27-Nor-5-cholestenoic acid behaves as an inverse agonist. This correlates with the capacity of the complex to better bind corepressors rather than coactivators. The C-25 methyl moiety would be necessary for the maintenance of a torsioned conformation of the steroid side chain that stabilizes an active LXRβ state. ConclusionWe found that a 27-nor analog is able to act as a LXR ligand. Interestingly, this minimal structural change on the steroid triggered a drastic change in the LXR response. General significanceResults contribute to improve our understanding on the molecular basis of LXRβ mechanisms of action and provide a new scaffold in the quest for selective LXR modulators.

Abstract 190: Serum HDL-C Decreases in MicroRNA-33b Knock-in Mice for an Intron of Sterol Regulatory Element-binding Factor 1 (Srebf1)

Background: MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33 (miR-33a) located within the intron of sterol regulatory element-binding factor (SREBF) 2 controls cholesterol homeostasis. Primates, but not rodents, express a second miR-33 gene (miR-33b) from an intron of SREBF1. To address miR-33b function in vivo, we developed humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron. Methods: The human miR-33b sequence was introduced into intron 16 of mouse Srebf1 because miR-33b is located in intron 16 of human SREBF1 and there are high homologies in exons 16 and 17 between human and mouse. The expression of serum miRNA was normalized with cel-miR-39 as a spike-in control. Results: We successfully established miR-33b knock-in (KI) mice with C57BL/6 background and this miR-33b KI strategy did not alter Srebf1 intron 16 splicing, which was confirmed by RT-PCR and sequencing. An LXR agonist T0901317, which induces Srebf1 expression, enhanced miR-33b expression in primary hepatocytes and the liver of miR-33 KI mice. The protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. Peritoneal macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Serum HDL-C levels were reduced by almost 35% in miR-33b KI mice. HPLC elution analysis showed that the decreased HDL-C levels were mainly composed of very large-, large-, medium sized HDL-C, which was compatible with the previous results of miR-33a deficient mice. Next, we measured miR-33b levels in serum of human. The expressions of miR-33b-3p were inversely correlated with serum HDL-C levels (P=0.025, R=0.336). Conclusions: miR-33b KI mice for an intron of Srebf1 showed reduced HDL-C level and serum miR-33b-3p levels were inversely correlated with HDL-C levels in human. These results indicate that miR-33b can be a potential target for raising HDL-C and may account for lower HDL-C levels in humans than those in mice. These mice will aid in elucidating the roles of miR-33s and screening of the drugs that can alter miR-33s levels and activities.

The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization.

BackgroundThe nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of “M2c” anti-inflammatory cells.MethodsWe cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils.ResultsWe found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206+ CD163+ CD16+) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206+ CD209+ CD163− MerTK−) to M2c (CD206high CD209− CD163+ MerTK+) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability.ConclusionsOur results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.

Development of novel sterol-based Liver X Receptor agonists as therapeutics for inflammatory diseases (HUM1P.307)

Abstract Liver X Receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR by synthetic agonists, including T0901317 and GW3965, attenuates progression of inflammatory diseases of animal models. However, the adverse effects of traditional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. The endogenous LXR agonist desmosterol and its sterol-based analog DMHCA activate LXR without the lipogenic action in the liver - this inspired us to develop anti-inflammatory therapies based on these analogs. Here, we show DMHCA oral treatment in mice significantly reduce DSS colitis-induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. This reduced inflammation is associated with induced LXR target gene expression, suggesting DMHCA attenuating colitis is mediated, at least partially, through LXR activation. In addition, DMHCA treatment dramatically suppresses inflammatory cytokine expression in traumatic brain injury model. Importantly, in both disease models, DMHCA does not increase liver lipid or serum ALT levels, while T0901317 results in significant liver lipid accumulation and injury. The anti-inflammatory property of DMHCA is recapitulated in vitro in CD3/CD28 activated human PBMCs. Overall, these results provide evidence for the development of sterol-based LXR agonists as novel therapeutics for inflammatory diseases.