LXRα Promoter Research Articles

Myristica fragrans promotes ABCA1 expression and cholesterol efflux in THP-1-derived macrophages.

Myristica fragrans is a traditional herbal medicine and has been shown to alleviate the development of atherosclerosis. However, the anti-atherogenic mechanisms of M. fragrans are still to be addressed. In this study, we explored the effect of M. fragrans on lipid metabolism and inflammation and its mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction and western blot analysis results showed that M. fragrans promotes cholesterol efflux from THP-1-derived macrophages and reduces intracellular total cholesterol, cholesterol ester, and free cholesterol contents in a dose- and a time-dependent manner. Further study found that liver X receptor alpha (LXRα) antagonist GGPP significantly blocked the upregulation of ABCA1 expression with M. fragrans treatment. In addition, chromatin immunoprecipitation assay confirmed that GATA binding protein 3 (GATA3) can bind to the LXRα promoter, and inhibition of GATA3 led to the downregulation of LXRα and ATP-binding cassette subfamily A member 1 expression. Furthermore, M. fragrans reduced lipid accumulation, followed by decreasing tumor necrosis factor-α, interleukin (IL)-6, and IL-1β and increasing IL-10 produced by THP-1-derived macrophages. Therefore, M. fragrans is identified as a valuable therapeutic medicine for atherosclerotic cardiovascular disease.

Decreased H3K9 acetylation level of LXRα mediated dexamethasone-induced placental cholesterol transport dysfunction

Due to the insufficient fetal cholesterol synthesis, maternal cholesterol transport through the placenta becomes an important source of fetal cholesterol pool, which is essential for fetal growth and development. This study aimed to investigate the effects of dexamethasone on fetal cholesterol levels, and explore its placental mechanism. Pregnant Wistar rats were injected subcutaneously with dexamethasone (0.8 mg/kg·d) from gestational day 9 to 20. Results showed that dexamethasone increased maternal serum total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) levels, as well as placental cholesterol synthesis and TC concentration, while reduced fetal birth weight, and serum TC, HDL-C and LDL-C levels. Meanwhile, the expression of placental cholesterol transporters, including low-density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-B1) and ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) were decreased by dexamethasone. Furthermore, the expression of glucocorticoid receptor (GR) and histone deacetylase 3 (HDAC3) were increased, while the H3K9ac and expression levels of liver X receptor α (LXRα) promoter were reduced. In human trophoblast cell line (BeWo), dexamethasone concentration-dependently decreased the expression levels of LDLR, SR-B1, ABCA1, ABCG1 as well as LXRα. Dexamethasone (2500 nM) induced GR translocation into nucleus and recruited HDAC3. Furthermore, LXRα agonist and GR inhibitor reversed respectively dexamethasone-induced the expression inhibitions of cholesterol transporter and LXRα, and HDAC3 siRNA reversed the H3K9ac level of LXRα promoter and its expression. Together, dexamethasone impaired placental cholesterol transport and eventually decreased fetal cholesterol levels, which is related to the down-regulation of LXRα mediated by GR/HDAC3/H3K9ac signaling.

The Liver X Receptor Is Upregulated in Monocyte-Derived Macrophages and Modulates Inflammatory Cytokines Based on LXRα Polymorphism.

Liver X receptors (LXRs) have emerged as important regulators of inflammatory gene expression. Previously, we had reported that an LXRα gene promoter polymorphism (-1830 T > C) is associated with systemic lupus erythematosus (SLE). Therefore, we assessed cytokine expression in relation to LXRα polymorphism in monocyte-derived macrophages from patients with SLE. Macrophages were obtained after 72 hours of culture of human monocytes supplemented with phorbol 12-myristate 13-acetate. Cells were transfected with LXRα promoter constructs. Additionally, peripheral blood mononuclear cell- (PBMC-) derived macrophages from the patients were evaluated for proinflammatory cytokines in relation to the genotypes of LXRα -1830 T > C. The expression of LXRα was increased in macrophages; levels of proinflammatory cytokines were decreased with LXRα expression. Production of proinflammatory cytokines varied depending on LXRα -1830 T > C genotype. In particular, expression of LXRα was decreased and that of proinflammatory cytokines was increased for LXRα -1830 TC genotype compared to that for TT genotype. The data were consistent in PBMC-derived macrophages from patients with SLE. Increased proinflammatory cytokines is related to TLR7 and TLR9 expression. These data suggest that the expression levels of LXRα, according to LXRα -1830 T > C genotype, may contribute to the inflammatory response by induction of inflammatory cytokines in SLE.

Maternal fructose induces gender-dependent changes in both LXRα promoter methylation and cholesterol metabolism in progeny

Fructose consumption from added sugars correlates with the epidemic rise in obesity, metabolic syndrome and cardiovascular diseases. However, consumption of beverages containing fructose is allowed during gestation. We have investigated whether maternal fructose intake produces subsequent changes in cholesterol metabolism of progeny. Carbohydrates were supplied to pregnant rats in drinking water (10% w/v solution) throughout gestation. Adult male and female descendants from fructose-fed, control or glucose-fed mothers were studied. Male offspring from fructose-fed mothers had elevated plasma HDL-cholesterol levels, whereas female progeny from fructose-fed mothers presented lower levels of non-HDL cholesterol vs. the other two groups. Liver X-receptor (LXR), an important regulator of cholesterol metabolism, and its target genes such as scavenger receptor B1, ATP-binding cassette (ABC)G5 and cholesterol 7-alpha hydroxylase showed decreased gene expression in males from fructose-fed mothers and the opposite in the female progeny. Moreover, the expression of a number of LXRα target genes related to lipogenesis paralleled to that for LXRα expression. In accordance with this, LXRα gene promoter methylation was increased in males from fructose-fed mothers and decreased in the corresponding group of females. Surprisingly, plasma folic acid levels, an important methyl-group donor, were augmented in males from fructose-fed mothers and diminished in female offspring. Maternal fructose intake produces a fetal programming that influences, in a gender-dependent manner, the transcription factor LXRα epigenetically, and both hepatic mRNA gene expression and plasma parameters of cholesterol metabolism in adult progeny. Changes in the LXRα promoter methylation might be related to the availability of the methyl donor folate.

Expression and methylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are associated with fatty liver syndrome in chicken

The typical characteristic of fatty liver syndrome (FLS) is an increased hepatic triacylglycerol content, and a sudden decline in egg production often occurs. FLS may develop into fatty liver hemorrhagic syndrome (FLHS), characterized by sudden death from hepatic rupture and hemorrhage. DNA methylation is associated with transcriptional silencing, leading to the etiology and pathogenesis of some animal diseases. The roles of DNA methylation in the genesis of FLS, however, are largely unknown. The lipogenic methyl-deficient diet (MDD) caused FLS similar to human nonalcoholic steatohepatitis (NASH). After 16 Jingxing-Huang (JXH) hens were fed MDD for 10 wk, eight exhibited FLS (designated as FLS-susceptible birds); the remainder, without FLS, served as controls (NFLS). Physiological and biochemical variables, gene expression levels, and DNA methylation were determined in the liver. The development of FLS in JXH hens was accompanied by abnormal lipid accumulation. Relative expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and microsomal triglyceride transfer protein (MTTP) were significantly up-regulated in the FLS group in comparison with the NFLS group. The transcript abundance of sterol regulatory element binding protein 1 (SREBP-1c), stearoyl-CoA desaturase (SCD), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor alpha (PPARα), and peroxisome proliferator-activated receptor gamma (PPARγ) did not differ between the two groups. Interestingly, MTTP and ACC mRNA abundance were negatively correlated with the level of promoter methylation. The extent of DNA methylation of the cytosine-guanine (CpG) sites in the SREBP-1c, FAS, PPARα, and LXRα promoter regions was also analyzed by direct sequencing but none differed between FLS and NFLS birds. Taken together, these results specify link DNA methylation to the pathogenesis of FLS in chickens.

Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5′ deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the −111 to −42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except −42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the −111/+384 LXRα promoter but not of the −42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the −50 to −40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.

HDAC3 interacts with sumoylated C/EBPα to negatively regulate the LXRα expression in rat hepatocytes

The expression changes of liver X receptor alpha (LXRα), histone deacetylase 3 (HDAC3) and CCAAT/enhancer binding protein alpha (C/EBPα) were detected in liver tissues of our high-fat-diet E3 rat model. The aim of this study is to pinpoint the molecular mechanism of HDAC3 and C/EBPα to orchestrate LXRα expression in hepatocytes. We confirmed that LXRα and its target genes were negatively regulated by HDAC3 in stable expressed clones with pEGFP-Hdac3 or shRNA-Hdac3 vector. However, transient pEGFP-C/EBPα plasmid transfection showed an upregulation of LXRα expression and C/EBPα enhanced LXRα promoter activity in a dose-dependent manner in CBRH-7919 cells. By using 5′-serial deletion reporter analysis, we identified that fragment from −2881 to −1181bp of LXRα promoter was responsible for C/EBPα binding to the promoter, especially CBS1 and CBS4 were identified essentially by using ChIP and luciferase reporter assay. Co-IP, qRT-PCR and ChIP revealed that HDAC3 interacted with C/EBPα co-regulated LXRα expression. Sumoylation of C/EBPα at lysine 159 was detected in CBRH-7919 cells with transient overexpressed C/EBPα, and Co-IP assay detected that sumoylated C/EBPα interacted with more HDAC3 than C/EBPα K159L mutant. Luciferase reporter assay demonstrated that C/EBPα participated in HDAC3-repressed LXRα transcription, and HDAC3 was involved in sumoylated C/EBPα-inactivated LXRα activity. Luciferase reporter assay demonstrated that sumoylation of C/EBPα by SUMO-1 directly reversed the activation of C/EBPα on LXRα promoter. The results suggested that HDAC3 interacts with sumoylated C/EBPα to negatively regulate the LXRα expression.

Elevated Expression of Liver X Receptor Alpha (LXRα) in Myocardium of Streptozotocin-Induced Diabetic Rats

The present study was designed to investigate the myocardial expression of liver X receptor alpha (LXRα) in a streptozotocin (STZ)-induced diabetic rat model. Immunohistochemical staining, quantitative real-time RT-PCR, and Western blot analysis were used to determine the expression of LXRα in the myocardium of STZ-induced diabetic rats. The myocardial expression of LXRα target genes, long-chain acyl-CoA synthetase 3 (ACSL3), fatty acid transporter protein (FAT/CD36), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 were also detected. Bisulfite sequencing analysis was employed to examine the methylation status of the CpG island at the LXRα promoter region in the myocardium of STZ-induced diabetic rats. We found that LXRα mRNA and protein expression in the left ventricles, right ventricles, and atria of diabetic rats were gradually increased during the progression of diabetic cardiomyopathy (DCM). The mRNA expression levels of ACSL3 and FAT/CD36 and the protein expression levels of ABCA1 and ABCG1 were also markedly increased in different heart chambers of diabetic rats. Moreover, there was a significant difference in the methylation status of LXRα gene between the ventricles of control and diabetic rats (P < 0.05). Our findings suggest that elevated expression of LXRα may be involved in the progression of DCM, and demethylation of LXRα is likely to be responsible for its increased expression in myocardial tissues.

Resveratrol regulates the expression of LXR-α in human macrophages

The naturally occurring polyphenol resveratrol has been associated with the beneficial effects of red wine consumption on cardiovascular disease and shown to inhibit atherosclerosis in animal models. To determine if resveratrol affects the expression of genes that control lipid homeostasis in human macrophages, we measured expression changes in the LXR-α pathway, crucial to cholesterol efflux, and in genes that mediate lipoprotein uptake. Resveratrol treatment of THP-1 macrophages induced LXR-α at mRNA and protein levels. Increased recruitment of RNA polymerase II to the LXR-α promoter suggested that up-regulation was at least partly mediated by transcriptional mechanisms. Resveratrol also induced LXR-α in human monocyte-derived macrophages together with elevated ABCA1 and ABCG1 mRNA levels. Moreover, resveratrol repressed the expression of the lipid uptake genes LPL and SR-AII. The ability of resveratrol to modulate expression of the genes involved in lipid uptake and efflux suggests that polyphenols can potentially limit cholesterol accumulation in human macrophages.

Glucocorticoid response and promoter occupancy of the mouse LXRα gene

The liver X receptors α and β (LXRα and LXRβ) are members of the nuclear receptor superfamily of proteins which are highly expressed in metabolically active tissues. They regulate gene expression of critical genes involved in cholesterol catabolism and transport, lipid and triglyceride biosynthesis, and carbohydrate metabolism in response to distinct oxysterol intermediates in the cholesterol metabolic pathway. Several LXR target genes have been identified, but there is limited information on how expression of the LXRs themselves is controlled. In this study we have characterized the upstream flanking region of the mouse LXRα gene. Transient transfections show that the LXRα promoter is able to drive transcription of a luciferase reporter gene, however, the transcriptional potential of the promoter in the cell lines used was low. The −2143 to −1513 region of the promoter mediates repression of reporter gene activity in all cells analyzed and multiple DNA–protein interactions were detected in this region by DNase I footprinting. The Zta, Ets, and Hes1 transcription factors were all shown to mediate alterations in reporter gene activity driven by LXRα promoter deletion constructs. These factors have been linked to cell cycle and differentiation processes suggesting that expression of LXRα might be under control of signalling mechanisms regulating cell proliferation. Several putative binding sites of the glucocorticoid receptor (GR) were identified in the LXRα promoter and transient cotransfections of the GR and LXRα promoter deletion constructs induced reporter gene activity. Addition of dexamethasone, a GR agonist, abolished this effect suggesting cross talk between GR and LXR signalling.

Potentiation of liver X receptor transcriptional activity by peroxisome-proliferator-activated receptor gamma co-activator 1 alpha.

Peroxisome-proliferator-activated receptor (PPAR) gamma co-activator 1 alpha (PGC-1 alpha/PPARGC1) plays an important role in energy metabolism by co-ordinating transcriptional programmes of mitochondrial biogenesis, adaptive thermogenesis and fatty acid beta-oxidation. PGC-1 alpha has also been identified to play a role in the intermediary metabolism by co-activating key transcription factors of hepatic gluconeogenesis and glucose uptake in muscles. In the present study, we show that PGC-1 alpha serves as a co-activator for the liver X receptor (LXR) alpha, known to contribute to the regulation of cellular cholesterol homoeostasis. In transient transfection studies, PGC-1 alpha amplified the LXR-mediated autoregulation of the LXR alpha promoter in a human brown adipocyte line and in 3T3-L1 cells via an LXR response element described previously. LXR-mediated transactivation via a natural LXR response element from the cholesteryl ester transfer-protein gene promoter was also enhanced by PGC-1 alpha in a ligand-dependent manner. Mutational analysis showed that the LXXLL signature motif (L2) of PGC-1 alpha was essential for co-activation of LXR-mediated transcriptional responses. This motif is located in the vicinity of the binding region for a putative repressor described previously. The repressor sequesters PGC-1 alpha from PPAR alpha and the glucocorticoid receptor, and this repressor did not interfere with PGC-1 alpha-mediated co-activation of LXR-dependent gene transcription. Moreover, inhibition of p38 mitogen-activated protein kinase signalling, shown to abolish the co-activation of PPAR alpha by PGC-1 alpha, had only a moderate inhibitory effect on the co-activation of LXR. These results identify PGC-1 alpha as a bona fide LXR co-activator and implicate distinct interfaces of PGC-1 alpha and/or additional cofactors in the modulation of LXR and PPAR alpha transcriptional activities.

Different regulation of the LXRα promoter activity by isoforms of CCAAT/enhancer-binding proteins

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRα promoter was thus studied to investigate if LXR α gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXR α gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRα promoter. C/EBPα upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPβ and C/EBPδ had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPα and C/EBPβ downregulated expression of the reporter gene while C/EBPδ induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRα promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRβ is constitutively expressed during the entire differentiation process while LXRα is induced upon addition of differentiation mix.

Members of the Nuclear Factor 1 Family Reduce the Transcriptional Potential of the Nuclear Receptor LXRα Promoter

Expression of the LXRα nuclear receptor in liver is predicted to affect cholesterol and lipid metabolism. Here we show that a short fragment from the LXRα gene promoter spanning the region from −144 to +43 relative to the mRNA initiation site can drive transcription of a reporter gene. Under basal conditions, in vitro DNase I footprinting demonstrated interaction between nuclear proteins and an NF1 recognition site in close vicinity to the transcriptional initiation. Both supershift, mutational analyses in EMSA and transfections provided evidence that the NF1 (nuclear factor I) transcription factor interacts with the LXRα promoter. All four members of the NF1 family were found to suppress the transcriptional activity indicating a general inhibitory effect on LXRα expression. A similar regulation by NF1 was also observed when using a fragment from the LXRα promoter extending up to position −3033 therefore giving the inhibitory effect of NF1 a significant impact on LXRα gene expression.

Autoregulation of the human liver X receptor alpha promoter.

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.