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Taurine, which is abundant in seafood, has antiatherogenic activities in both animals and humans; however, its molecular target has been elusive. We examined whether taurine could activate liver X receptor-α (LXR-α), a critical transcription factor in the regulation of reverse cholesterol transport in macrophages. Taurine bound directly to LXR-α in a reporter gene assay, time-resolved fluorescence resonance energy transfer analysis, and limited protease digestion experiment. Macrophage cells incubated with taurine showed reduced cellular cholesterol and induced medium cholesterol in a dose-dependent manner with the induction of ATP-binding cassette transporter A1 and G gene and protein expression. In hepatocytes, taurine significantly induced Insig-2a levels and delayed nuclear translocation of the sterol regulatory element-binding protein 1 (SREBP-1) protein, resulting in a dose-dependent reduction in the cellular lipid levels without inducing the expression of fatty acid synthesis genes. Taurine is a direct LXR-α ligand, represses cholesterol accumulation, and modulates the expression of genes involved in reverse cholesterol transport in macrophages, without inducing hepatic lipogenesis. The induction of Insig-2a suppressed the nuclear translocation of SREBP-1c. Show less

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or Δ-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). 'Transport' was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by >2-fold 1 h post H37Rv infection. By 1 h post Δ-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by >2-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection. Show less

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, β and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner. Show less

Panax notoginseng (Burk.) F.H. Chen has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, and Korea. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. The purpose of this study was to investigate whether Panax notoginseng saponins (PNS) attenuated atherosclerosis by inducing liver X receptor alpha (LXRα) expression and to elucidate the mechanisms responsible for the effects. The AS rats were treated once daily with PNS (100 mg/kg, i.p.), and pathological changes in the aorta were observed using Sudan IV staining. The expression of LXRα in the aortic wall was measured by Western blot analysis. THP-1 macrophages were cultured with PNS in the presence or absence of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRα antagonist. The expression of LXRα and its target genes ATP-binding cassette A1 and G1 (ABCA1, ABCG1) were determined by qRT-PCR. The transcriptional activation of the LXRα gene promoter was analyzed by a reporter assay. The NF-κB DNA binding activity and the expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) was evaluated respectively by Trans-AM NF-κB ELISA and ELISA in THP-1 macrophages that were stimulated with LPS after treatment with PNS and GGPP. PNS treatment alleviated the typical pathological changes associated with atherosclerosis in rats. The expression of LXRα was increased in rat aortas after treatment with PNS. In vitro, PNS increased LXRα mRNA levels in THP-1 macrophages. The reporter assays showed that PNS enhanced transcriptional activation of the LXRα gene promoter and led to the upregulation of ABCA1 and ABCG1 expression. This upregulation could be reversed by treatment with GGPP. Additionally, PNS inhibited NF-κB DNA binding activity and reduced secretion of IL-6 and MCP-1 in LPS-stimulated THP-1 macrophages. These effects could be reversed by GGPP. The results indicated that the PNS-mediated attenuation of AS may, at least partly, due to LXRα uprergulation. The mechanisms of action included enhancement transcriptional activation of the LXRα gene promoter by PNS and subsequent upregulation of ABCA1 and ABCG1 and inhibition of NF-κB DNA binding activity. Show less

Selenium neutralizes interleukin-1β (IL-1β) induced inflammatory responses in chondrocytes. We investigated potential mechanisms for this through in vitro knock down of three major selenoproteins, Iodothyronine Deiodinase-2 (DIO2), Glutathione Peroxidase-1 (GPX1), and Thioredoxin Reductase-1 (TR1) in primary human chondrocytes. Primary human chondrocytes were transfected with scrambled small interfering ribonucleic acid (siRNA) or siRNA specific for DIO2, GPX1 and TR1. After 48 h, transfected cells were cultured in serum free media for 48 h, with or without 10 pg/ml IL-1β for the final 24h. The efficiency of siRNAs was confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot analysis. The gene expression, by qRT-PCR, of cyclooxygenase-2 (COX2), IL-1β, and Liver X receptor (LXR) alpha and beta was evaluated to determine the impact of selenoprotein knockdown on inflammatory responses in chondrocytes. The messenger RNA (mRNA) expression of DIO2, GPX1, and TR1 was significantly decreased by the specific siRNAs (reduced 56%, P=0.0004; 96%, P<0.0001; and 66%, P<0.0001, respectively). Suppression of DIO2, but not GPX1 or TR1, significantly increased (~2-fold) both basal (P=0.0005) and IL-1β induced (P<0.0001) COX2 gene expression. Similarly, suppression of DIO2 significantly increased (∼9-fold) IL-1β induced IL-1β gene expression (P=0.0056) and resulted in a 32% (P=0.0044) decrease in LXRα gene expression but no effect on LXRβ. Suppression of the selenoprotein DIO2 resulted in strong pro-inflammatory effects with increased expression of inflammatory mediators, IL-1β and COX2, and decreased expression of LXRα suggesting that this may be the upstream target through which the anti-inflammatory effects of DIO2 are mediated. Show less

Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice. Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice. Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells. Show less

Molecular mechanisms contributing to hepatitis C virus (HCV)-associated steatosis are not well established, although HCV gene expression has been shown to alter host cell cholesterol/lipid metabolism. As liver X receptors (LXRs) play a role as key modulators of metabolism signaling in the development of steatosis, we aimed to investigate in an HCV in vitro model the effect of HCV NS5A protein, core protein, and viral replication on the intracellular lipid accumulation and the LXRα-regulated expression of lipogenic genes. The effects of LXRα siRNA or agonist GW3965 treatment on lipogenesis and HCV replication capacity in our HCV replicon system were also examined. NS5A- and core-expressing cells and replicon-containing cells exhibited an increase of lipid accumulation by inducing the gene expression and the transcriptional activity of LXRα, and leading to an increased expression of its lipogenic target genes sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor-γ, and fatty acid synthase. Transcriptional induction by NS5A protein, core protein, and viral replication occurred via LXR response element activation in the lipogenic gene promoter. No physical association between HCV proteins and LXRα was observed, whereas NS5A and core proteins indirectly upregulated LXRα through the phosphatidylinositol 3-kinase pathway. Finally, it was found that LXRα knockdown or agonist-mediated LXRα induction directly regulated HCV-induced lipogenesis and HCV replication efficiency in replicon-containing cells. Combined, our data suggest that LXRα-mediated regulation of lipogenesis by core and NS5A proteins may contribute to HCV-induced liver steatosis and to the efficient replication of HCV. Show less

The liver X receptor α (LXRα) is a nuclear receptor of the transcription factor and is known to play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis in humans and rodents. However, very little information is available on the role of LXRα in the regulation of fatty acid synthesis in the goat mammary gland. In this investigation, a cDNA was isolated from the mammary gland of Xinong Saanen dairy goats and designated as goat LXRα. RT-PCR and RACE gave rise to the full-length cDNA of LXRα, which was comprised of 1654 bp and characterized by an ORF of 1344 bp and 5'- and 3'-UTR regions of 150 and 160 bp, respectively. The deduced amino acid sequence encodes 477 amino acids with a predicted molecular weight (MW) of 50.4kDa and a theoretical isoelectric point (pI) of 6.3. Additionally, homology search and sequence multi-alignment indicated that the putative goat LXRα amino acid sequence is very similar to those of cattle, mice, rats, swine, and humans. Bioinformatic predictions demonstrated that the LXRα protein is located in the nucleus, containing characteristic signatures of a nuclear receptor with DNA-binding domain (DBD) and ligand-binding domain (LBD). Real-time quantitative PCR suggested that LXRα was predominantly expressed in the small intestine, liver, spleen and mammary gland. Treatment of goat mammary gland epithelial cells (GMEC) with different concentrations (i.e., 0.01, 0.1, 1 μM) of T0901317, a synthetic agonist of LXRα, resulted in elevated sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) mRNA levels in response to LXRα activation. The association between different T0901317 concentrations and fatty acid composition in GMEC also was examined using gas chromatography (GC). The results showed that activation of LXRα significantly increased GMEC C18:1 and C18:2 contents, but did not affect levels of saturated fatty acids (SFA). These discoveries are consistent with the notion that LXRα plays a key role in controlling lipogenesis and regulating synthesis of unsaturated fatty acids (UFA) in the mammary gland of goats, which may prove useful in regulation of milk fat production. Show less

Both inflammation and cholesterol accumulation play important roles in the development of non-alcoholic fatty liver disease. This study was undertaken to investigate whether inflammation aggravated cholesterol accumulation via disrupting hepatic cholesterol export and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, and tumor necrosis factor alpha (TNF-α) stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammation. Intracellular cholesterol level was examined by Oil Red O staining and quantitative analysis. Bile acid level was quantified by colorimetric analysis. (3)[H] cholesterol assay by scintillation counting was performed to evaluate the cholesterol efflux. The mRNA and protein expression was examined by real-time polymerase chain reaction and Western blot. Inflammation increased cholesterol accumulation in livers of C57BL/6J mice and in HepG2 cells. High-fat diet in mice and low-density lipoprotein (LDL) loading in HepG2 cells increased bile acid synthesis and cholesterol efflux, enhanced the mRNA and protein expression of liver X receptor α (LXRα), peroxisome proliferator-activated receptors (PPARα, γ), cholesterol 7α-hydroxylase (CYP7A1) and ATP-binding cassette transporter A1 (ABCA1). However, inflammation reduced bile acid synthesis and cholesterol efflux even in high-fat-diet-fed mice and HepG2 cells in the presence of LDL loading. The enhanced effects of these genes and proteins expression due to high-fat diet and LDL loading were inhibited by inflammation both in vivo and in vitro. Inflammation disrupted PPAR-LXR-CYP7A1/ABCA1-mediated bile acid synthesis and cholesterol efflux resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells. Show less

Resveratrol (RSV), a naturally occurring polyphenolic stilbenoid, is known to possess potent anti-atherogenic properties; however, the effect of RSV on hypercholesterolemia is not fully understood. We hypothesized that RSV decreases blood cholesterol levels through the activation of cholesterol 7α-hydroxylase (CYP7A1)-mediated bile acid synthetic pathway pathways in vitro and in vivo. In this study, we evaluated body weight, serum lipid concentrations, hepatic lipid content and the size of the bile acid pool in high-fat diet (HFD)-fed C57BL/6 J mice that were treated with RSV. In addition, we characterized the underlying mechanism of the effects of RSV in HepG2 hepatocytes by Western blot analysis. RSV (200 mg/kg per day) reduced body weight and liver weight gains, improved serum lipid parameters, reduced hepatic cholesterol accumulation and increased the bile acid pool size in mice fed an HFD for 8 wks. RSV significantly increased liver expression of CYP7A1 mRNA and protein and CYP7A1 enzyme activity. Furthermore, RSV treatment upregulated CYP7A1 expression and induced liver X receptor alpha (LXRα) activation in a time- and dose-dependent manner in HepG2 cells. In addition, the specific liver X receptor alpha (LXRα) inhibitor geranylgeranyl pyrophosphate (GGPP) inhibited the RSV-induced expression of CYP7A1 in HepG2 hepatocytes. The beneficial effects of RSV on HFD-induced hypercholesterolemia are mediated through LXRα signaling pathways, suggesting a potential target for the prevention of dyslipidemia. Show less

The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells. Show less

This study is a post-hoc analysis of a subset of patients who participated in our multi-institutional case-control study that evaluated the effects of pitavastatin in patients with non-alcoholic fatty liver disease (NAFLD) with hypercholesterolemia. Serum samples of fifteen patients with biopsy-proven NAFLD with dyslipidemia were investigated. Serum markers of lipid metabolism were quantified by liquid chromatography-mass spectrometry (LC-MS)/MS. These data were then compared with those of 36 sex- and age-matched healthy controls. In addition, changes in these markers produced by treatment with pitavastatin were evaluated. Serum non-cholesterol sterols, reflecting intestinal cholesterol absorption, were significantly lower in the NAFLD patients compared to the controls, and the cholesterol synthesis marker, the ratio of lathosterol to cholesterol, was not significantly different between the two groups. Serum proportions of liver X receptor α (LXRα) ligand oxysterols (ratios to cholesterol) were significantly elevated in the NAFLD patients compared to the controls. The sum of oxysterols relative to cholesterol and the homeostasis model assessment as an index of insulin resistance (HOMA-IR) were significantly correlated. The marker representing cholesterol synthesis was significantly suppressed by pitavastatin treatment, from 3 months after initiation of the treatment, and the suppression remained significant during the observation period. The markers representing cholesterol absorption were unchanged at 3 months, but had significantly increased at 12 months. Serum oxysterol levels relative to cholesterol maintained high values and did not change significantly during the 12-month period of treatment. We speculate that serum LXRα ligand oxysterol levels (relative to cholesterol) could be surrogate markers of insulin resistance, and that high oxysterol levels in the circulation may play an important role in the development of hepatic and peripheral insulin resistance followed by NAFLD. Show less

Benign prostatic hyperplasia is a nonmalignant enlargement of the prostate that commonly occurs in older men. We show that liver X receptor (Lxr)-α knockout mice (lxrα(-/-)) develop ventral prostate hypertrophy, correlating with an overaccumulation of secreted proteins in prostatic ducts and an alteration of vesicular trafficking in epithelial cells. In the fluid of the lxrα(-/-) prostates, spermine binding protein is highly accumulated and shows a 3000-fold increase of its mRNA. This overexpression is mediated by androgen hypersensitivity in lxrα(-/-) mice, restricted to the ventral prostate. Generation of chimeric recombinant prostates demonstrates that Lxrα is involved in the establishment of the epithelial-mesenchymal interactions in the mouse prostate. Altogether these results point out the crucial role of Lxrα in the homeostasis of the ventral prostate and suggest lxrα(-/-) mice may be a good model to investigate the molecular mechanisms of benign prostatic hyperplasia. Show less

Nuclear receptors (NRs) are master regulators of transcriptional programs that integrate the homeostatic control of almost all biological processes. Their direct mode of ligand regulation and genome interaction is at the core of modern pharmacology. The two liver X receptors LXRα and LXRβ are among the emerging newer drug targets within the NR family. LXRs are best known as nuclear oxysterol receptors and physiological regulators of lipid and cholesterol metabolism that also act in an anti-inflammatory way. Because LXRs control diverse pathways in development, reproduction, metabolism, immunity and inflammation, they have potential as therapeutic targets for diseases as diverse as lipid disorders, atherosclerosis, chronic inflammation, autoimmunity, cancer and neurodegenerative diseases. Recent insights into LXR signaling suggest future targeting strategies aiming at increasing LXR subtype and pathway selectivity. This review discusses the current status of our understanding of LXR biology and pharmacology, with an emphasis on the molecular aspects of LXR signaling that constitute the potential of LXRs as drug targets. Show less

Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 μmol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar (3)H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXRα) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXRα-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXRα activation in human enterocytes. Exogenous LXRα agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXRα-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXRα-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXRα antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol. Show less

Pregnancy-associated plasma protein-A (PAPP-A) has been involved in the atherosclerotic process through regulation of local expression of IGF-1 that mediates the activation of the phosphatidylinositol-3 (PI3-K) and Akt kinase (Akt) signaling cascades which lead to constitutive nitric oxide formation, with its attending vasodilator, antiplatelet and insulin-sensitizing actions. In addition, IGF-1 may decreased cholesterol efflux through reductions of expression in ABCA1 and SR-B1 by the PI3-K/Akt signaling pathway. In the current study, we examined whether PAPP-A was involved in LXRα regulation and in expression of ABCA1, ABCG1 or SR-B1 through the IGF-I-mediated signaling pathway (IGF/PI3-K/Akt). Results showed that PAPP-A significantly decreased expression of ABCA1, ABCG1 and SR-BI at both transcriptional and translational levels in a dose-dependent and time-dependent manner. Cellular cholesterol content was increased while cholesterol efflux was decreased by PAPP-A treatment. Moreover, LXRα which can regulate the expression of ABCA1, ABCG1 and SR-B1, was also down-regulated by PAPP-A treatment. LXRα-specific activation by LXRα agonist almost rescued the down-regulation of ABCA1, ABCG1 and SR-B1 expression by PAPP-A. In addition, PAPP-A can induce the IGF-1/PI3-K/Akt pathway in macrophages. Furthermore, our results indicate that the decreased levels observed in LXRα, ABCA1, ABCG1 and SR-B1 mRNA and protein levels upon treating cells with PAPP-A were strongly impaired with the PI3-K inhibitors or IGF-1R siRNA while the MAPK cascade inhibitor did not execute this effect, indicating that the process of ABCA1, ABCG1 and SR-BI degradation by PAPP-A involves the IGF-1/PI3-K/Akt pathway. In conclusion, PAPP-A may first down-regulate expression of LXRα through the IGF-1/PI3-K/Akt signaling pathway and then decrease expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells. Therefore, our study provided one of the mechanisms for understanding the critical effect of PAPP-A in pathogenesis of atherosclerosis. Show less

Interleukin (IL)-18 and IL-12 synergize for the production of interferon (IFN)-γ, which can downregulate ATP-binding cassette transporter A1 (ABCA1) expression. The aim of the present study was to investigate the effect of IL-18 and/or IL-12 on ABCA1 expression. IL-18 combined with IL-12 decreased ABCA1 expression and cellular cholesterol efflux in THP-1 macrophage-derived foam cells, whereas IL-18 or IL-12 alone had no effect. IL-12 increased IL-18 receptor (IL-18R) expression, which was suppressed by small interfering RNA (siRNA) for signal transducer and activator of transcription 3. IL-18R but not IL-12 receptor siRNA completely reversed the effects of IL-18 and IL-12 on ABCA1 expression and cellular cholesterol efflux. Treatment with IL-18 plus IL-12 markedly augmented nuclear translocation of nuclear factor (NF)-κB but had no effect on expression and activity of liver X receptor α. IL-18 and IL-12 also significantly increased zinc finger protein 202 (ZNF202) levels and IFN-γ secretion. Furthermore, siRNA for ZNF202 or IFN-γ significantly impaired IL-18/IL-12-induced suppression of ABCA1, whereas NF-κB siRNA treatment blocked IL-18/IL-12' action on ZNF202 levels, IFN-γ secretion, and ABCA1 expression. IL-18 and IL-12 together can decrease ABCA1 expression and cellular cholesterol efflux in THP-1 macrophage-derived foam cells through the IL-18R/NF-κB signaling pathway. Show less

Although previous studies have shown that consumption of anthocyanin extract from plant foods reduces hypercholesterolemia and the severity of atherosclerosis in different animal models, the mechanisms of these actions remained unclear. This study investigated whether pure anthocyanin inhibit atherosclerosis development and reduce hypercholesterolemia in the apolipoprotein E (ApoE)-deficient mice through enhancement of fecal bile acid excretion, a critical pathway for eliminating circulation cholesterol from the body. Five-week-old male ApoE-deficient mice were fed the AIN-93G diet supplemented with or without cyanidin-3-O-β-glucoside (0.06% w/w) for 12 weeks. Results showed that cyanidin-3-O-β-glucoside consumption inhibited the formation of aortic sinus plaque and reduced hypercholesterolemia, along with promoted fecal bile acid excretion and upregulated hepatic cholesterol 7a-hydroxylase expression (CYP7A1). In mouse primary hepatocytes, cyanidin-3-O-β-glucoside treatment increased bile acid synthesis and CYP7A1 expression in a liver X receptor alpha (LXRα)-)-dependent manner. Scintillation proximity and time-resolved fluorescence resonance energy transfer assays revealed that cyanidin-3-O-β-glucoside functions as an agonist of LXRα. Our results indicate that the hypocholesterolemic activity of cyanidin-3-O-β-glucoside was, at least in part, mediated by activating the potential LXRα-CYP7A1-bile acid excretion pathway, thus contributing to the antiatherogenic effect of cyanidin-3-O-β-glucoside. Importantly, cyanidin-3-O-β-glucoside could activate LXRα in an agonist-dependent manner. Show less

Pharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated. We investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317. HDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1 week treatment. T0901317 treatment induced fecal neutral sterol secretion by ~300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body. Induction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion. Show less

GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. Show less

Oxysterols/oxysterol receptors have been shown to modulate several immune cell subsets, such as macrophages, T-cells and B-cells, neutrophils and dendritic cells (DCs). They participate in the control of several pathologic processes, that is, infectious diseases, atherosclerosis and autoimmunity. Moreover, some oxysterols have also been shown to favor tumor progression by dampening the antitumor immune response. The cellular responses generated by oxysterols depend on the engagement of Liver X Receptor (LXR) α and/or β isoforms, which induce activation of target genes or trans-repression of pro-inflammatory gene transcription. Recently, some reports have described a different mechanism of action of oxysterols, mediated by the engagement of G-Protein Coupled Receptors. Here, we summarize LXR-dependent and LXR-independent responses of oxysterols on immune cells with possible effects on tumor development. Show less

The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE(-/-) background, deletion of LXRα, but not LXRβ, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRβ on the ApoE(-/-) background led to an even more severe cholesterol accumulation phenotype compared to LXRα(-/-)ApoE(-/-) mice, indicating that LXRβ does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα(-/-)ApoE(-/-) and LXRβ(-/-)ApoE(-/-) mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα(-/-)ApoE(-/-) mice. Mechanistically, this defect was linked to a specific requirement for LXRα(-/-) in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice. Show less

ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR). Since ABCA1 plays a pivotal role in cholesterol and HDL metabolism, identification of a compound capable of increasing its expression may be beneficial for the prevention and therapy of atherosclerosis. Firefly luciferase reporter assays were developed for human ABCA1 promoters and LXR enhancers, and an in-house phytochemical library was screened. It was found that a citrus flavonoid, hesperetin (1), increased ABCA1 promoter and LXR enhancer activities in THP-1 macrophages. It was also found that this flavonoid promoted PPAR-enhancing activity. In accordance with these findings, 1 increased mRNA and protein expression of ABCA1 and consequently upregulated ApoA-I-mediated cholesterol efflux. These results provide evidence that 1 promotes ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression through the activation of LXRα and PPARγ. Show less

The activity of 6-phosphofructo-1-kinase is strictly controlled by fructose-2,6-bisphosphate, the level of which is regulated by another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2). PFK2/FBP2 is a bifunctional enzyme, having kinase and phosphatase activities, and regulates both glycolysis and gluconeogenesis. Here, we examined the hormonal regulation of the PFK2/FBP2 gene in vitro using the reporter assay, the electromobility shift assay (EMSA), and the chromatin immunoprecipitation (ChIP) assay in HuH7 cells and also using the mouse liver in vivo. We found that the transcriptional activity of the PFK2/FBP2 gene was stimulated by insulin and inhibited by cAMP and glucocorticoid. Liver X receptor (LXR) α showed a potent and specific stimulatory effect on PFK2/FBP2 gene transcription. Deletion and mutagenesis analyses identified the LXR response element (LXRE) in the 5'-promoter region of the PFK2/FBP2 gene. Binding of LXRα was confirmed by the EMSA and ChIP assay. Endogenous PFK2/FBP2 mRNA in the mouse liver was increased in the fasting/refeeding state compared with the fasting state. Altogether, PFK2/FBP2 gene transcription is found to be regulated in a way that is more similar to other glycolytic enzyme genes than to gluconeogenic genes. Furthermore, our data strongly suggest that LXRα is one of the key regulators of PFK2/FBP2 gene transcription. Show less

In our previous study, we demonstrated that lycopene can inhibit the proliferation of androgen-dependent prostate LNCaP cancer cells through the activation of the peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor alpha (LXRα)-ATP-binding cassette transporter 1 (ABCA1) pathway. However, it is still unclear whether lycopene possesses similar effects in androgen-independent prostate cancer cells DU145 and PC-3. As lycopene inhibited the proliferation of both cell types to a similar extent, we chose DU145 cells for most of the subsequent studies. We show that lycopene significantly increased protein and mRNA expression of PPARγ, LXRα and ABCA1 and cholesterol efflux (i.e., decreased cellular cholesterol and increased cholesterol in culture medium). Lycopene (10 μM) in the presence of a specific antagonist of PPARγ (GW9662) or of LXRα (GGPP) restored the proliferation of DU145 cells and significantly suppressed lycopene-induced protein and mRNA expression of PPARγ and LXRα and cholesterol efflux. Liver X receptor α knockdown by siRNA against LXRα significantly promoted the proliferation of DU145 cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. Furthermore, lycopene in combination with the LXRα agonist T0901317 exhibited synergistic effects on cell proliferation and protein expression of PPARγ, LXRα and ABCA1. These results demonstrate that lycopene can inhibit DU145 cell proliferation via PPARγ-LXRα-ABCA1 pathway and that lycopene and T0901317 exhibit synergistic effects. Show less

To investigate the role of liver X receptors (LXRs) on endothelin-1 (ET-1) induced murine HL-1 cardiomyocytes hypertrophy. Cultured murine HL-1 cardiomyocytes were divided into four experiment groups: (1) CONTROL GROUP:treated with DMSO; (2) T0901317 group:treated with LXRs agonist T0901317 (1 µmol/L); (3) ET-1 group:treated with ET-1 (1 nmol/L); (4) T0901317 + ET-1 group:treated with T0901317 (1 µmol/L) for 8 hours, then treated with ET-1 (1 nmol/L). Twenty-four hours later, immunofluorescent staining was performed on HL-1 cells, the surface area of HL-1 cells was analyzed with NIH Image J software, and the synthetic rate of protein in HL-1 cells was detected by (3)H-leucine incorporation. The mRNA level of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MyHC) was measured by quantitative realtime PCR. The effect of T0901317 on mRNA expression of ANP was also detected after LXRs gene silencing. The surface area of HL-1 cells, mRNA expression of ANP and β-MyHC, and (3)H-leucine incorporation in ET-1 group were 2.00 ± 0.29, 1.98 ± 0.47, 2.13 ± 0.39 and 1.79 ± 0.17, respectively, which were significantly higher than those of control group (1.00 ± 0.26, 1.00 ± 0.21, 1.00 ± 0.31 and 1.00 ± 0.03, respectively, all P < 0.05). Compared with ET-1 group, the surface area of HL-1 cells, mRNA expression of ANP and β-MyHC, and (3)H-leucine incorporation were significantly decreased in T0901317 + ET-1 group (1.24 ± 0.25, 1.19 ± 0.21, 1.48 ± 0.27 and 1.15 ± 0.11, respectively, all P < 0.05). After inhibition of LXRα/β expression in HL-1 cardiomyocytes using the specific siRNAs, the mRNA expression of ANP in T0901317 + ET-1 group was 1.78 ± 0.05, which was similar as that in ET-1 group (1.94 ± 0.17, P > 0.05). T0901317, an agonist of LXRs, could inhibit ET-1 induced cardiac hypertrophy in vitro, and LXR ligand-mediated inhibition on ANP mRNA expression by T0901317 is receptor dependent. Show less

Structural modification of a series of dual LXRα/β agonists led to the identification of a new class of LXRβ partial agonists. An X-ray co-crystal structure shows that a representative member of this series, pyrrole 5, binds to LXRβ with a reversed orientation compared to 1. Show less

The liver X receptor α (LXRα) is a nuclear receptor that is involved in regulation of lipid metabolism, cellular proliferation and apoptosis, and immunity. In this report, we characterize three human LXRα isoforms with variation in the ligand-binding domain (LBD). While examining the expression of LXRα3, which lacks 60 amino acids within the LBD, we identified two novel transcripts that encode LXRα-LBD variants (LXRα4 and LXRα5). LXRα4 has an insertion of 64 amino acids in helix 4/5, and LXRα5 lacks the C-terminal helices 7 to 12 due to a termination codon in an additional exon that encodes an intron in the LXRα1 mRNA. LXRα3, LXRα4, and LXRα5 were expressed at lower levels compared with LXRα1 in many human tissues and cell lines. We also observed weak expression of LXRα3 and LXRα4 in several tissues of mice. LXR ligand treatment induced differential regulation of LXRα isoform mRNA expression in a cell type-dependent manner. Whereas LXRα3 had no effect, LXRα4 has weak transactivation, retinoid X receptor (RXR) heterodimerization, and coactivator recruitment activities. LXRα5 interacted with a corepressor in a ligand-independent manner and inhibited LXRα1 transactivation and target gene expression when overexpressed. Combination of LXRα5 cotransfection and LXRα antagonist treatment produced additive effects on the inhibition of ligand-dependent LXRα1 activation. We constructed structural models of the LXRα4-LBD and its complexes with ligand, RXR-LBD, and coactivator peptide. The models showed that the insertion in the LBD can be predicted to disrupt RXR heterodimerization. Regulation of LXRα pre-mRNA splicing may be involved in the pathogenesis of LXRα-related diseases. Show less

This study investigated the impact of chronic exposure to continuous (CoP4) versus cyclic progesterone (CyP4) alone or in combination with 17β-estradiol (E2) on gene expression profiles targeting bioenergetics, metabolism and inflammation in the adult female rat hippocampus. High-throughput qRT-PCR analyses revealed that ovarian hormonal depletion induced by ovariectomy (OVX) led to multiple significant gene expression alterations, which were to a great extent reversed by co-administration of E2 and CyP4. In contrast, co-administration of E2 and CoP4 induced a pattern highly resembling OVX. Bioinformatics analyses further revealed clear disparities in functional profiles associated with E2+CoP4 and E2+CyP4. Genes involved in mitochondrial energy (ATP synthase α subunit; Atp5a1), redox homeostasis (peroxiredoxin 5; Prdx5), insulin signaling (insulin-like growth factor I; Igf1), and cholesterol trafficking (liver X receptor α subtype; Nr1h3), differed in direction of regulation by E2+CoP4 (down-regulation relative to OVX) and E2+CyP4 (up-regulation relative to OVX). In contrast, genes involved in amyloid metabolism (β-secretase; Bace1) differed only in degree of regulation, as both E2+CoP4 and E2+CyP4 induced down-regulation at different efficacy. E2+CyP4-induced changes could be associated with regulation of progesterone receptor membrane component 1(Pgrmc1). In summary, results from this study provide evidence at the molecular level that differing regimens of hormone therapy (HT) can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause, would imply that the common regimen of continuous combined HT may have adverse consequences whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function throughout menopausal aging. Show less

Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRβ, with overlapping but not identical functions, we investigated whether LXRα and LXRβ exert different effects on intestinal cholesterol absorption. Wild-type (WT), LXRα(-/-) and LXRβ(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965. When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRβ(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. In the intestine, LXRα and LXRβ seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRβ may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRβ-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway. Show less