LXR Ligand Research Articles

Abstract 3336: Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells

Abstract Liver X Receptors (LXRs) are nuclear receptors that act as ligand-modulated transcription factors. LXRs are overexpressed in various cancers, including pancreatic cancer, and targeting LXR with small molecule ligands has emerged as a promising therapeutic strategy in cancer. Current small molecule LXR ligands were originally developed for the treatment of atherosclerosis and other metabolic diseases. To identify ligands specifically targeting cancer cells, we screened a focused library of drug-like molecules predicted to bind to LXR using molecular docking. In the screen, novel LXR ligand GAC0001E5 (1E5) exhibited significant anti-proliferative effects in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Functionally, 1E5 is an LXR inverse agonist which decreases LXR target gene expression and a “degrader” of LXR proteins following prolonged treatment. We posit that this novel ligand inhibits PDAC cells by downregulating the expression of LXR target genes. To test this hypothesis, we treated cancer cells with the previously described LXR inverse agonist SR9238 and examined its effect on cancer cell proliferation. Interestingly, treatments with SR9238 had no effect on pancreatic cancer cell proliferation as compared to the novel ligand 1E5 and the 1E5 derivative KD-95, suggesting that LXR inverse agonism was insufficient to inhibit cell proliferation. To determine whether SR9238 has similar effects on LXR protein levels as novel ligands, we performed western analysis of protein expression following ligand treatment. In contrast to the novel ligands 1E5 and KD-95, treatments with SR9238 increased LXR protein levels. Furthermore, time-course studies in cycloheximide treated cells indicated that novel ligands decreased protein stability whereas SR9238 had the opposite effect. Inhibition of proteasomal degradation and autophagy abolished the effects of 1E5 and KD-95 in a cell line-dependent manner. Related to these observations, previously published data revealed that knockdown of LXR expression greatly diminished PDAC cell proliferation. Taken together, these findings indicate that LXR expression is essential in pancreatic cancer cells and suggest that the novel ligands may disrupt its involvement in protein-protein interactions and non-genomic mechanisms. Citation Format: Abhinav Bagchi, Kasuni T. Gamage, Scott R. Gilbertson, Chin-Yo Lin. Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3336.

Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

Ouabagenin, an aglycone of cardiotonic steroid ouabain, functions as LXR ligand but avoids the increase in the SREBP-1 by inducing Krüppel-like factor 15

Ouabagenin, an aglycone of cardiotonic steroid ouabain, functions as LXR ligand but avoids the increase in the SREBP-1 by inducing Krüppel-like factor 15

Nanoparticle-based “Two-pronged” approach to regress atherosclerosis by simultaneous modulation of cholesterol influx and efflux

Reduction of lipoprotein uptake by macrophages and stimulation of cholesterol efflux are two essential steps required for atherosclerotic plaque regression. We used the optimized mannose-functionalized dendrimeric nanoparticle (mDNP)-based platform for macrophage-specific delivery of therapeutics to simultaneously deliver SR-A siRNA (to reduce LDL uptake) and LXR ligand (LXR-L, to stimulate cholesterol efflux) – a novel “Two-pronged” approach to facilitate plaque regression. mDNP-mediated delivery of SR-A siRNA led to a significant reduction in SR-A expression with a corresponding decrease in uptake of oxLDL. Delivery of LXR-L increased expression of ABCA1/G1 and cholesterol efflux. Combined delivery of siRNA and LXR-L led to a significantly greater decrease in macrophage cholesterol content compared to either treatment alone. Administration of this in vitro optimized formulation of mDNP complexed with SR-A-siRNA and LXR-L (Two-pronged complex) to atherosclerotic LDLR−/− mice fed western diet (TD88137) led to significant regression of atherosclerotic plaques with a corresponding decrease in aortic cholesterol content.

Combination of MEK1/2 inhibitor and LXR ligand synergistically inhibit atherosclerosis in LDLR deficient mice

Combined LXR ligand (T0901317) and MEK1/2 inhibitor (U0126) not only reduces atherosclerosis in apoE deficient mice, but also blocks LXR ligand-induced fatty liver and hypertriglyceridemia. However, the atheroprotective function of combined T0901317 and U0126 should be further investigated in LDLR deficient (LDLR-/-) mice since deficiency of LDLR not apoE can occur to humans with a high frequency. Herein, we validated the effectiveness of this combinational therapy on the development of atherosclerosis in LDLR-/- mice to demonstrate its potential application in clinic. We found although T0901317 or U0126 alone reduced atherosclerotic plaques in en face and aortic root areas in HFD-fed LDLR-/- mice, their combination inhibited lesions in a synergistic manner. Combined U0126 and T0901317 had no effect on serum total cholesterol levels. T0901317 deceased HDL-cholesterol levels, which was restored by combined U0126. Meanwhile, U0126 alleviated T0901317-induced triglyceride accumulation, the major adverse effect of T0901317 which limits its clinical utility. Mechanistically, U0126 reduced fatty acid de novo synthesis by inhibiting hepatic fatty acid synthase (FASN) expression, thereby correcting T0901317-induced triglyceride overproduction. In conclusion, our study demonstrates that combination of MEK1/2 inhibitor and LXR ligand can synergistically reduce atherosclerosis in LDLR deficient mice without lipogenic side effects.

P728Subclinical atherosclerosis and its progression are modulated by perilipin-2 through a feed-forward loop between LXR and autophagy

Abstract Background Hyperlipidemia is a major risk factor for cardiovascular disease and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin-2 reduces plasma triglycerides and may therefore be beneficial for atherosclerosis development. Purpose We sought to delineate putative beneficial effects of the Pro251 variant of perlipin-2 on subclinical atherosclerosis and the mechanism by which it acts. Methods A pan-European cohort of high-risk individuals where carotid intima-media thickness has been assessed was adopted. Human primary monocyte-derived macrophages were prepared from whole blood from individuals recruited by perilipin-2 genotype, or from buffy coats from the our University hospital blood central. Results The Pro251 variant of perilipin-2 is associated with decreased intima-media thickness at baseline and 30 months follow-up. Using human primary monocyte-derived macrophages from carriers of the beneficial Pro251 variant we show that this variant increases autophagy activity, cholesterol efflux, and a controlled inflammatory response. Through extensive mechanistic studies we demonstrate that increase in autophagy activity is accompanied with an increase in LXR activity and that LXR and autophagy reciprocally activate each other in a feed-forward loop, regulated by CYP27A1 and 27OH-cholesterol. Conclusions For the first time, we show that perilipin-2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin-2 modulates levels of the LXR ligand 27OH-cholesterol and initiates a feed-forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin-2 exerts its beneficial effects on subclinical atherosclerosis. Acknowledgement/Funding The Swedish Research Council, Swedish Heart-Lung Foundation, Marianne and Marcus Wallenberg's Foundation, Swedish Medical Society

24(S),25-Epoxycholesterol and cholesterol 24S-hydroxylase (CYP46A1) overexpression promote midbrain dopaminergic neurogenesis in vivo

The liver X receptors Lxrα/NR1H3 and Lxrβ/NR1H2 are ligand-dependent nuclear receptors critical for midbrain dopaminergic (mDA) neuron development. We found previously that 24(S),25-epoxycholesterol (24,25-EC), the most potent and abundant Lxr ligand in the developing mouse midbrain, promotes mDA neurogenesis in vitro. In this study, we demonstrate that 24,25-EC promotes mDA neurogenesis in an Lxr-dependent manner in the developing mouse midbrain in vivo and also prevents toxicity induced by the Lxr inhibitor geranylgeranyl pyrophosphate. Furthermore, using MS, we show that overexpression of human cholesterol 24S-hydroxylase (CYP46A1) increases the levels of both 24(S)-hydroxycholesterol (24-HC) and 24,25-EC in the developing midbrain, resulting in a specific increase in mDA neurogenesis in vitro and in vivo, but has no effect on oculomotor or red nucleus neurogenesis. 24-HC, unlike 24,25-EC, did not affect in vitro neurogenesis, indicating that the neurogenic effect of 24,25-EC on mDA neurons is specific. Combined, our results indicate that increased levels of 24,25-EC in vivo, by intracerebroventricular delivery in WT mice or by overexpression of its biosynthetic enzyme CYP46A1, specifically promote mDA neurogenesis. We propose that increasing the levels of 24,25-EC in vivo may be a useful strategy to combat the loss of mDA neurons in Parkinson's disease.

Akt/mTOR and AMPK signaling pathways are responsible for liver X receptor agonist GW3965-enhanced gefitinib sensitivity in non-small cell lung cancer cell lines.

BackgroundThis study was to systemically analyze the mechanism of LXR ligand GW3965-induced sensitivity to EGFR-TKI in EGFR-TKI-resistant non-small cell lung cancer (NSCLC) cell lines.MethodsGefitinib-resistant PC9 cell line (EGFR exon 19 deletion) was treated with single and combined treatment with GW3965 and gefitinib. Cell viability, apoptosis and autophagy were detected using MTT, flow cytometric analysis and immunofluorescent analysis, respectively. Autophagy-related signaling pathways were detected using Western blot analysis.ResultsInhibited cell viability by single and combined treatment with gefitinib and GW3965 were observed. Combined treatment with gefitinib and GW3965 increased LC3 II/I ratio and Beclin 1 expression. Synergistic effect of gefitinib and GW3965 on apoptosis and autophagosome accumulation as well as on the inhibition of Akt/mTOR signaling and activation of AMP-activated protein kinase (AMPK) was observed in gefitinib-resistant PC9 cells. AMPK expression showed similar profile with apoptosis and autophagy of PC9 cells.ConclusionsWe confirmed that GW3965 and gefitinib showed synergistic effect on Akt/mTOR inhibition, apoptosis and autophagy of lung cancer cells. Gefitinib sensitivity in PC9 cell line might be mediated by Akt/mTOR, AMPK and JNK signaling pathways.

Akt/mTOR and AMPK signaling pathways are responsible for liver X receptor agonist GW3965-enhanced gefitinib sensitivity in nonsmall cell lung cancer cell lines

Background: This study was to systemically analyze the mechanism of LXR ligand GW3965-induced sensitivity to EGFR-TKI in EGFR-TKI-resistant non-small cell lung cancer (NSCLC) cell lines. Methods: Gefitinib-resistant PC9 cell line (EGFR exon 19 deletion) was treated with single and combined treatment with GW3965 and gefitinib. Cell viability, apoptosis and autophagy were detected using MTT, flow cytometric analysis and immunofluorescent analysis, respectively. Autophagy-related signaling pathways were detected using Western blot analysis. Results: Inhibited cell viability by single and combined treatment with gefitinib and GW3965 were observed. Combined treatment with gefitinib and GW3965 increased LC3 II/I ratio and Beclin 1 expression. Synergistic effect of gefitinib and GW3965 on apoptosis and autophagosome accumulation as well as on the inhibition of Akt/mTOR signaling and activation of AMP-activated protein kinase (AMPK) was observed in gefitinib-resistant PC9 cells. AMPK expression showed similar profile with apoptosis and autophagy of PC9 cells. Conclusions: We confirmed that GW3965 and gefitinib showed synergistic effect on Akt/mTOR inhibition, apoptosis and autophagy of lung cancer cells. Gefitinib sensitivity in PC9 cell line might be mediated by Akt/mTOR, AMPK and JNK signaling pathways.

Nuclear receptor ligands induce TREM-1 expression on dendritic cells: analysis of their role in tumors

ABSTRACTDendritic cells (DCs) initiate adaptive immune responses after their migration to secondary lymphoid organs. The LXR ligands/oxysterols and the RXR ligand 9-cis Retinoic Acid (9-cis RA) were shown to dampen DC migration to lymphoid organs through the inhibition of CCR7 expression. We performed transcriptomics of DCs undergoing maturation in the presence of the LXR ligand 22R-Hydroxycholesterol (22R-HC). The analysis highlighted more than 1500 genes modulated by 22R-HC treatment, including the triggering receptor expressed on myeloid cells (TREM)-1, which was found markedly up-regulated. We tested the effect of other nuclear receptor ligands (NRL) and we reported the induction of TREM-1 following RXR, RAR and VDR activation. From a functional point of view, triggering of TREM-1 induced by retinoids increased TNFα and IL-1β release, suggesting an active role of NRL-activated TREM-1+ DCs in inflammation-driven diseases, including cancer. Consistently with this hypothesis we detected DCs expressing TREM-1 in pleural effusions and ascites of cancer patients, an observation validated by the induction of TREM-1, LXR and RAR target genes when monocyte-DCs were activated in the presence of tumor-conditioned fluids. Finally, we observed a better control of LLC tumor growth in Trem-1−/- bone marrow chimera mice as compared to wild type chimera mice. Future studies will be necessary to shed light on the mechanism of TREM-1 induction by distinct NRL, and to characterize the role of TREM-1+ DCs in tumor growth.

Abstract 036: Functionalized Nanoparticle Based “Two-pronged” Approach to Attenuate/regress Atherosclerosis by Simultaneous Knock Down OfSR-A and LXR-mediated Stimulation of Macrophage Cholesterol Efflux

Imbalance between lipoprotein uptake and cholesterol efflux from macrophages underlies the formation of foam cells and development of fatty streaks, a process that starts early in life. While currently available lipid lowering interventions attenuate the progression of atherosclerotic plaques, no targeted therapy is presently available for reduction of existing plaques, a process dependent not only on limiting further accumulation of cholesterol but also on stimulation of cholesterol efflux. We earlier reported the development of a mannose functionalized dendrimeric nanoparticles (mDNP)-based platform for targeted delivery of therapeutics to arterial plaque associated macrophages. In this study, we used the optimized mDNP platform to simultaneously deliver SR-A siRNA (to knock down SR-A and limit modified LDL uptake) and LXR ligand (LXR-L, to stimulate macrophage cholesterol efflux by inducing expression of ABCA1/G1) - a “ Two Pronged ” approach. Compared to non-specific NS-siRNA, mDNP mediated delivery of SR-A siRNA led to significant reduction in expression of SR-A (A&B) with a corresponding decrease in uptake of DiI-labeled oxLDL (C). Delivery of LXR-L increased expression of ABCA1/G1 (D) as well as cholesterol efflux (E) and simultaneous delivery of SR-A siRNA did not alter these LXR-L effects. However, combined delivery of siRNA and LXR-L led to significantly higher decrease in macrophage cholesterol content compared to either treatment alone (F). Administration of this in vitro optimized formulation of mDNP complexed with SR-A siRNA and LXR-L to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to a dramatic reduction in lesion area compared to single treatment alone (G). In conclusion, targeted delivery of multiple therapeutics using mDNP platform represents a novel strategy to not only reduce lesion size but it can also be used to simultaneously alter many lesion characteristics such as reducing inflammation or enhancing efferocytosis.

Fat-specific protein 27 is a novel target gene of liver X receptor α.

Fat-specific protein 27 (FSP27) is highly expressed in the fatty liver of genetically obese ob/ob mice and promotes hepatic triglyceride (TG) accumulation. The nuclear hormone receptor liver X receptor α (LXRα) also plays a critical role in the control of TG levels in the liver. The present study demonstrated transcriptional regulation of Fsp27a and Fsp27b genes by LXRα. Treatment with the LXR ligand T0901317 markedly increased Fsp27a and Fsp27b mRNAs in wild-type C57BL/6J and ob/ob mouse livers. A reporter assay indicated that two LXR-responsive elements (LXREs) are necessary for LXRα-dependent induction of Fsp27a and Fsp27b promoter activities. Furthermore, the LXRα/retinoid X receptor α complex is capable of directly binding to the two LXREs both invitro and invivo. These results suggest that LXRα positively regulates Fsp27a and Fsp27b expression through two functional LXREs. Fsp27a/b are novel LXR target genes in the ob/ob fatty liver.

Amelioration of amyloid β-induced retinal inflammatory responses by a LXR agonist TO901317 is associated with inhibition of the NF-κB signaling and NLRP3 inflammasome

Amyloid β (Aβ) is a pathogenic peptide associated with many neurodegenerative diseases such as Alzheimer’s disease and Parkinson's disease. The retinal inflammation in response to Aβ is implicated in the pathogenesis of several ocular diseases including age-related macular degeneration, Alzheimer's-related optic neuropathy and glaucoma. In the present study, we found that a single intravitreal injection of oligomeric Aβ1-40 in mouse activated the NLRP3 inflammasome and the NF-κB signaling, induced the production of inflammatory cytokines including TNF-α and IL-6. In addition, Aβ1-40 caused retinal function impairment while no noticeable morphological changes were observed under light microscope. Furthermore, immunohistochemical results showed that Aβ1-40 enhanced the number of Iba1-positive cells in the inner retina. The mRNA expressions of LXRα and LXRβ decreased in the neuroretina of the Aβ1-40-injected mice. No significant difference was found on the protein expressions of LXRs and ABCA1 in both neuroretina and RPE/choroid complex between the Aβ1-40-injected group and the control group. A synthetic LXR ligand, TO901317 (TO90), enhanced the expressions of LXRα and ABCA1 at both mRNA and protein levels in the Aβ1-40-injected mice, while the LXRβ expression was unchanged. TO90 preserved ERG a- and b-wave amplitudes and reduced the number of Iba1-positive cells in the Aβ1-40-treated retina. Furthermore, TO90 down-regulated the mRNA levels of TNF-α and IL-6, as well as the expressions of p-IκBα, NLRP3, caspase-1 and IL-1β in the Aβ1-40-injected animals. We suggest that activation of LXRα and its target gene ABCA1 exerts potent anti-inflammatory effect on the Aβ-treated retina.

Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice.

We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ−/−, C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production.

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.

An LXR-NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux.

LXR-cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3-LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux.

Purple Perilla frutescens extracts enhanced cholesterol efflux and inhibited ER stress in macrophages exposed to oxidized LDL (831.8)

There is growing evidence that excess lipids can stimulate endoplasmic reticulum (ER) stress in atherosclerosis. However, the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. The current study tested that oxidized LDL promoted ER stress in macrophages, which was blocked by purple Perilla frutescens extracts (PPE). The ER stressor tunicamycin was employed as a positive control. Treating oxidized LDL for 24 h accumulated lipid droplets in macrophages and formed foam cells, evidenced by Oil red O staining. PPE suppressed foam cell formation, but enhanced ATP‐binding cassette (ABC) transporter A1 and G1, cholesterol efflux markers. Oxidized LDL as well as tunicamycin induced the ER stress markers of activating transcription factor 6 and this effect was blocked by 蠅5 μg/ml PPE, indicating that intracellular toxic lipids in macrophages initiated ER stress. PPE also down‐regulated the induction of the ER resident chaperone BiP/Grp78. The LXR ligand TO‐0131917 increased ABCA1 expression that was blunted by the ER stressor. Accordingly, an upstream mechanism that links toxic lipids to ER stress may entail the action of lipid chaperones through inhibition of LXR. Purple Perilla frutescens extract (PPE) is an annual herb of mint family, which has possessed against inflammation and cancer. Therefore, PPE enhanced cholesterol efflux and inhibited ER stress in macrophages exposed to lipotoxic signals associated with atherosclerosis.Grant Funding Source: Supported by Ministry for Food, Agriculture, Forestry and Fisheries and Brain Korea 21 plus

A Liver-X-Receptor Ligand, T0901317, Attenuates IgE Production and Airway Remodeling in Chronic Asthma Model of Mice

The liver-X-receptors have shown anti-inflammatory ability in several animal models of respiratory disease. Our purpose is to investigate the effect of LXR ligand in allergen-induced airway remodeling in mice. Ovalbumin-sensitized mice were chronically challenged with aerosolized ovalbumin for 8 weeks. Some mice were administered a LXR agonist, T0901317 (12.5, 25, 50 mg/kg bodyweight) before challenge. Then mice were evaluated for airway inflammation, airway hyperresponsiveness and airway remodeling. T0901317 failed to attenuate the inflammatory cells and Th2 cytokines in bronchoalveolar lavage fluid. But the application of T0901317 reduced the thickness of airway smooth muscle and the collagen deposition. Meanwhile, T0901317 treatment evidently abolished the high level of OVA-specific IgE, TGF-β1 and MMP-9 in lung. So LXRs may attenuate the progressing of airway remodeling, providing a potential treatment of asthma.

Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism

IntroductionLiver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment.MethodsTo further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments.ResultsWe observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival.ConclusionsTaken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.

179 NOVEL LXRALPHA CROSSTALK WITH THE INTERFERON REGULATORY FACTOR 8 MODULATES MACROPHAGES INFLAMMATORY PATHWAYS

The Liver X Receptors or LXRs are important modulators of cholesterol homeostasis and immune pathways. Accordingly, LXR activation by synthetic ligands decreases the progression of metabolic diseases with a strong inflammatory component such as atherosclerosis. Typically, LXRs control gene expression by heterodimerizing with the retinoid X receptor (RXR) and binding to LXR response elements or LXREs. We have now uncovered an additional mode of positive target gene regulation by LXR in macrophages that involves the hematopoietic transcription factors PU.1 and interferon regulatory factor 8 (IRF8), which control the development and function of several immune cell types. We previously demonstrated that levels of arginase 1, a metabolic enzyme with immunomodulatory actions, are induced by LXR in cultured macrophages and within atherosclerotic lesions undergoing regression. Moreover, elevated levels of arginase 1 are associated with the regression of atherosclerosis. We have also established that LXR does not regulate arginase 1 expression by directly binding to an LXRE in the arginase 1 gene, but instead induces the levels of IRF8 and promotes the interaction between IRF8 and PU.1 and their binding to the arginase 1 promoter. We have now evidence that in addition to arginase 1, the expression of other LXR target genes are dependent on IRF8 in macrophages. Microarray analysis was performed in differentiated bone marrow-derived macrophages (BMDM) activated with the specific LXR ligand GW3965 or vehicle. To examine whether the ligand response of identified LXR activated genes was dependent on IRF8 expression, IRF8 was selectively knocked down using RNAi. Results obtained were further validated in BMDM from IRF8-deficient mice. Interestingly, one of the IRF8-dependent LXR target genes we have identified this way encodes IL-18 binding protein (IL-18BP), a well-characterized endogenous inhibitor of cytokine signaling that has already been shown to modulate atherosclerosis. Il-18BP induced mRNA and protein expression by LXR ligands is dependent on LXR as demonstrated using LXR-deficient mice. Moreover, LXR ligands induce the promoter activity of IL-18BP as assessed by luciferase reporter assays. However, while LXR does not bind to the IL-18BP promoter, IRF8 binding to this locus is significantly enhanced by LXR ligand activation in BMDM as assessed by chromatin immunoprecipitation assays. In addition, to investigate the mechanism by which LXR ligands modulate IRF8 binding activity, LXR regulation of IRF8 post-translational modifications known to modulate its DNA binding was examined. Ligand-induced LXR enhances IRF8 phosphorylation in macrophages thus providing a potential mechanism underlying the induced IRF8 binding to LXR target genes. Collectively, this work identifies a novel crosstalk between IRF8 and LXR on the control of LXR macrophage gene expression that is likely to be relevant to the development of atherosclerosis.