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The biochemical and biological properties of 4β-hydroxycholesterol and of its isomer, 4α-hydroxycholesterol, are not well known. So, we determined the ability of 4α- and 4β-hydroxycholesterol to react with LXRα and LXRβ, and we characterized the activities of these oxysterols on oligodendrocytes which are myelin synthesizing cells. The effects of 4α- and 4β-hydroxycholesterol were studied on 158N murine oligodendrocytes to assess their activities on cell growth and viability, oxidative and inflammatory status. To this end different parameters were used: cell counting with trypan blue; identification of dead cells and cell cycle analysis with propidium iodide; evaluation of mitochondrial depolarization, lysosomal membrane integrity, actin depolimerization, nuclear morphology, and superoxide anion production after staining with JC-1, acridine orange, rhodamine-phalloidin, Hoechst 33342, and dihydroethidium, respectively; evaluation of ultrastructural changes by transmission electron microscopy, and cytokine quantification with a cytometric bead array. Only 4β-hydroxycholesterol is a LXRα and β agonist. No cytotoxic effects were found with 4α-hydroxycholesterol except a slight inhibition of cell growth at elevated concentrations. At high concentrations, 4β-hydroxycholesterol was not only able to inhibit cell growth, but also to induce cell death associated with a loss of mitochondrial transmembrane potential, dysfunctions of lysosomal membrane integrity, and superoxide anion overproduction. These side effects were lower than those observed with 7-ketocholesterol and 25-hydroxycholesterol used as positive controls. On oligodendrocyte murine primary cultures, only lysosomal membrane integrity was slightly affected under treatment with 4α- and 4β-hydroxycholesterol. So, 4α- and 4β-hydroxycholesterol have different biological activities. Their ability to induce cytotoxic effects on oligodendrocytes can be considered as weak comparatively to 7-ketocholesterol and 25-hydroxycholesterol. Show less

The role of cholesterol in female reproductive physiology has been suspected for a long time, while the molecular bases were unknown. Cholesterol is the precursor of ovarian steroid biosynthesis and is also essential for fertility. In the uterus, cholesterol is essential to achieve correct contractions at term, but an excessive uterine cholesterol concentration has been associated with contractility defects. Liver X Receptor (LXR) α and LXR β are nuclear receptors activated by oxysterols, oxidized derivatives of cholesterol. Since their discovery, the role of LXR in the control of cholesterol homeostasis has been widely described. Beyond their cholesterol-lowering role, more recent data have linked these nuclear receptors to various physiological processes. In particular, they control ovarian endocrine and exocrine functions, as well as uterine contractility. Their contribution to female reproductive cancers will also be discussed. This review will try to enlighten on the LXR as a molecular link between dietary cholesterol and reproductive diseases in women. In the future, a better comprehension of the various physiological processes regulated by the LXR will help to develop new ligands to prevent or to cure these pathologies in women. Show less

Emerging experimental and human evidence has linked altered hepatic cholesterol homeostasis and free cholesterol (FC) accumulation to the pathogenesis of non-alcoholic steatohepatits (NASH). This review focuses on cellular mechanisms of cholesterol toxicity involved in liver injury and on alterations in cholesterol homeostasis promoting hepatic cholesterol overload in NASH. FC accumulation injures hepatocytes directly, by disrupting mitochondrial and endoplasmic reticulum (ER) membrane integrity, triggering mitochondrial oxidative injury and ER stress, and by promoting generation of toxic oxysterols, and indirectly, by inducing adipose tissue dysfunction. Accumulation of oxidized LDL particles may also activate Kupffer and hepatic stellate cells, promoting liver inflammation and fibrogenesis. Hepatic cholesterol accumulation is driven by a deeply deranged cellular cholesterol homeostasis, characterized by elevated cholesterol synthesis and uptake from circulating lipoproteins and by a reduced cholesterol excretion. Extensive dysregulation of cellular cholesterol homeostasis by nuclear transcription factors sterol regulatory binding protein (SREBP)-2, liver X-receptor (LXR)-α and farnesoid X receptor (FXR) plays a key role in hepatic cholesterol accumulation in NASH. The therapeutic implications and opportunities for normalizing cellular cholesterol homeostasis in these patients are also discussed. Show less

Lipopolysaccharide (LPS), the principal component of Gram-negative bacterial cell walls, is a stimulator of osteoclastogenesis and thus a key factor in inflammatory bone loss. We have recently reported that the important cholesterol and inflammatory regulator, liver X receptor (LXRα/β), can potently inhibit osteoclast formation from bone marrow-derived osteoclast precursors in a bacterial/LPS environment. In this manuscript, we further studied the effect of the LXR agonist GW3965 on osteoclast differentiation in RAW264.7 promyelocytic leukemia cells exposed to LPS. We found that not only did activation of the LXR potently inhibit the formation of TRAP-positive osteoclast-like cells, but promoted a population of TRAP-negative mononuclear cells with high phagocytic activity. We observed reduced expression of the osteoclast markers TRAP/Acp5, Ctsk, Calcr and Oscar after 3-4days of GW3965 treatment, coinciding with an increase in the expression of the anti-osteoclastogenic factor Irf8. Expression of the macrophage/phagocytic marker Cd68 was increased, however the "classical" macrophage markers F4/80 and Cd14 and the "alternatively" activated macrophage markers Tgfβ and Il10 were not altered. Further, activation of LXR increased the expression of the macrophage survival gene AIM/SPα, a known LXR target gene, and osteoclast/macrophage-related markers (Mitf, Pu.1, Usf1/2, Ostm1 and Mfr). Although Akt phosphorylation was reduced, GW3965 seemed to act independently of MAPKs (p38, ERK, JNK) and NFκB, and had no inhibitory effect on cytokine expression (Tnfα, Il6, or Il1β). Our results indicate that activation of the LXR not only inhibits the differentiation of osteoclast-like cells from RAW264.7 cells in a bacterial/LPS environment, but is also involved in the fate determination of myeloid progenitor cells into macrophages with high phagocytic capacity. Show less

Oltipraz, a cancer chemopreventive agent, has an anti-steatotic effect via liver X receptor-α (LXRα) inhibition. Here we have assessed the biological activity of a major metabolite of oltipraz (M2) against liver steatosis and steatohepatitis and the underlying mechanism(s). Blood biochemistry and histopathology were assessed in high-fat diet (HFD)-fed mice treated with M2. An in vitroHepG2 cell model was used to study the mechanism of action. Immunoblotting, real-time PCR and luciferase reporter assays were performed to measure target protein or gene expression levels. M2 treatment inhibited HFD-induced steatohepatitis and diminished oxidative stress in liver. It increased expression of genes encoding proteins involved in mitochondrial fuel oxidation. Mitochondrial DNA content and oxygen consumption rate were enhanced. Moreover, M2 treatment repressed activity of LXRα and induction of its target genes, indicating anti-lipogenic effects. M2 activated AMP-activated protein kinase (AMPK). Inhibition of AMPK by over-expression of dominant negative AMPK (DN-AMPK) or by Compound C prevented M2 from inducing genes for fatty acid oxidation and repressed sterol regulatory element binding protein-1c (SREBP-1c) expression. M2 activated liver kinase B1 (LKB1) and increased the AMP/ATP ratio. LKB1 knockdown failed to reverse target protein modulations or AMPK activation by M2, supporting the proposal that both LKB1 and increased AMP/ATP ratio contribute to its anti-steatotic effect. M2 inhibited liver steatosis and steatohepatitis by enhancing mitochondrial fuel oxidation and inhibiting lipogenesis. These effects reflected activation of AMPK elicited by increases in LKB1 activity and AMP/ATP ratio. Show less

Bacterial-induced bone diseases, such as periodontitis and osteomyelitis, are chronic inflammatory diseases characterized by increased bone destruction as a result of enhanced osteoclastogenesis. The LXRα and -β are important modulators of inflammatory signaling and can potently inhibit RANKL-induced osteoclast differentiation. Here, we investigated the effects of the LXR agonist GW3965 on LPS-induced osteoclast differentiation. Mouse BMMs primed with RANKL for 24 h, then exposed to LPS in the presence of GW3965 for 4 days, formed significantly fewer and smaller TRAP(+)-multinucleated osteoclasts with reduced expression of osteoclast markers (Acp5, Ctsk, Mmp-9, Dc-stamp, and Itgβ3), along with inhibition of actin ring development. GW3965 was able to repress proinflammatory cytokine (TNF-α, IL-1β, IL-6, and IL-12p40) expression in BMMs exposed to LPS alone; however, once BMMs entered the osteoclast lineage following RANKL priming, GW3965 no longer inhibited cytokine expression. The inhibitory action of GW3965 involved the Akt pathway but seemed to be independent of MAPKs (p38, ERK, JNK) and NF-κB signaling. GW3965 acted in a LXRβ-dependent mechanism, as osteoclast differentiation was not inhibited in BMMs derived from LXRβ-/- mice. Finally, activation of LXR also inhibited differentiation in LPS-exposed mouse RAW264.7 cells. In conclusion, GW3965 acts through LXRβ to potently inhibit osteoclast differentiation from RANKL-primed BMMs in a LPS environment. In this respect, activation of the LXR could have a beneficial, therapeutic effect in the prevention of bacterial-induced bone erosion. Show less

Endocrine disrupting chemicals (EDCs) have emerged as a major public health issue because of their potentially disruptive effects on physiological hormonal actions. SXR (steroid xenobiotic receptor), also known as NR1I2, regulates CYP3A expression in response to exogenous chemicals, such as EDCs, after binding to SXRE (SXR response element). In our study, luciferase assay showed that 14 out of 55 EDCs could enhance SXR-mediated rat or human CYP3A gene transcription nearly evenly, and could also activate rat CYP7A1 gene transcription by cross-interaction of SXR and LXRE (LXRα response element). SXR diffused in the nucleus without ligand, whereas intranuclear foci of liganded SXR were produced. Furthermore, endogenous mRNA expression of CYP3A4 gene was enhanced by the 14 positive EDCs. Our results suggested a probable mechanism of EDCs disrupting the steroid or xenobiotic metabolism homeostasis via SXR. Show less

The oxysterol receptors LXRα and LXRβ are members of the nuclear receptor family and established transcriptional regulators of lipid metabolism with additional anti-inflammatory functions. Recent investigations have indicated an important role of LXRs in the control of proliferation. Here we further extend this knowledge to human colon cancer cells and proliferation in mouse colon. We show that activation of LXRs leads to a robust cell cycle arrest in colorectal adenocarcinoma cell lines. At the molecular level LXRs control expression of several cell cycle genes including Skp2, c-Myc, CDKs, cyclins, and p15. Furthermore, activation of LXRs causes hypo-phosphorylation of the retinoblastoma (Rb) tumor suppressor protein. Experiments performed in vivo show that the colon structure appears to be intact in LXR null mice. However, LXRαβ(-/-) mice show a significant increase of proliferation markers in colon compared to wild type mice and administration of the LXR specific agonist, GW3965 significantly reduced expression of proliferation in mouse colon. Taken together, these findings point toward a strong anti-proliferative effect of LXRs in colon revealing the potential of LXR ligands as possible anti cancer agents. Show less

Alzheimer's disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42. Show less

Both L-4F, one apolipoprotein A-1 mimetic peptide, and statins can reduce progression of atherosclerosis by different mechanisms. The combination of the two drugs can cause lesion regression by rendering HDL anti-inflammatory. We postulated that combination of L-4F and simvastatin may stimulate cholesterol efflux and related proteins expressions to alleviate atherosclerosis. Thirty male wild-type (W-T) C57 BL/6 mice and apo E(-/-) mice were divided into five groups: W-T group, atherosclerosis (AS) group, simvastatin group, L-4F group and the combination of simvastatin and L-4F group. After 16 weeks, serum lipids, atherosclerotic lesion areas, cholesterol efflux and the expressions of related proteins including ABCA1, SR-BI, ABCG1, LXRα and PPARγ were evaluated. The aortic atherosclerotic lesion areas were reduced more significantly by combination of both drugs than single agent, and cholesterol efflux was promoted more in combination group than simvastatin and L-4F group. Besides, the combination group promoted expressions of cholesterol efflux related proteins. The combination of L-4F and simvastatin reduced atherosclerotic lesions, which stimulates cholesterol efflux by promoting the expressions of related proteins. In addition, these results help us further understand that the regression of the atherosclerosis would be assessed by reduction in LDL-C with increase of cholesterol efflux. Show less

Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin. Show less

Fatty acid composition of meat is becoming more important due to consumer demand for high quality and healthy foods. The present study evaluated the associations of five candidate genes (FABP4, FASN, NR1H3, GH and SCD) with fatty acid composition in Korean cattle (Hanwoo). The g.3691G > A single nucleotide polymorphism (SNP) in the FABP4 gene had significant effects on high myristic acid (C14:0; P < 0.01) and palmitic (C16:0; P < 0.05) in animals having the GG genotype, and high arachidonic acid (C20:4; P < 0.05) in the AA genotype of Hanwoo. The FASN SNP at position g.17924G > A was also significantly associated with myristic acid (P < 0.01). In case of the SCD gene, a significant effect was only observed in myristoleic acid (C14:1; P < 0.01). However, SNPs in GH and NR1H3 genes showed no effects on fatty acid composition. The results indicate that SNPs in three candidate genes, FABP4, FASN and SCD, may be influential in breeding design for fatty acid composition in Hanwoo. Show less

Reverse cholesterol transport (RCT), a process to deliver excess cholesterol from the periphery to the liver for excretion from body, is a major atheroprotective property of high-density lipoproteins. As major transporters for cholesterol efflux in macrophages, ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) are critical for RCT. We investigated mechanisms for the regulation of ABCA1 and ABCG1 expression by fatty acids (FA) in RAW264.7 macrophages. Cells were incubated with 100 μmol/L of palmitic, oleic, linoleic, linolenic or eicosapentaenoic acids in the absence or presence of T0901317, a liver X receptor (LXR) agonist. Unsaturated FA, but not saturated FA, significantly reduced ABCA1 and ABCG1 mRNA without the agonist. Trichostatin A (TSA), a histone deacetylase inhibitor, not only increased basal ABC transporter expression but abrogated the transcriptional repression by unsaturated FA. The increased basal ABCA1 and ABCG1 mRNA by TSA paralleled the increased peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α expression, whereas LXRα and PGC-1β expression was significantly lowered. Although the repressive effect of ABCA1 and ABCG1 mRNA by unsaturated FA was abolished by T0901317, protein levels remained diminished. Chemical and genetic deficiency of protein kinase C δ did not abolish the repressive effect of linoleic acid on ABCA1 and ABCG1. In conclusion, unsaturated FA repressed ABCA1 and ABCG1 expression by two distinct mechanisms in RAW 264.7 macrophages: LXR-dependent transcriptional repression possibly by modulating histone acetylation state and LXR-independent posttranslational inhibition. Show less

Apolipoprotein M (ApoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in the plasma. Statins have effects on many HDL-associated apolipoproteins. However, it is unknown whether statins have effects on ApoM. In the present study, we investigated the effects of simvastatin on ApoM expression and the underlying mechanism(s). Simvastatin up-regulated hepatic ApoM mRNA and protein expression in mice. In HepG2 cells, simvastatin significantly enhanced ApoM mRNA and protein expression in a dose-dependent manner. Simvastatin increased hepatic hepatocyte nuclear factor-1α (HNF-1α) mRNA and reduced liver X receptor-α (LXRα) mRNA expression in mice. The simvastatin-induced up-regulation of ApoM was blocked by an HNF-1α inhibitor (UCDA) or an LXRα agonist (TO901317) in HepG2 cells which indicates that this effect is mediated via the regulation of HNF-1α and LXRα. In conclusion, simvastatin significantly up-regulated ApoM expression in vivo and in vitro, which indicates that ApoM is another novel apolipoprotein regulated by simvastatin. The mechanism of this effect is related to the regulation of HNF-1α and LXRα. Show less

The liver X receptor, LXRα, is an important regulator of genes involved in metabolism and inflammation. The mechanism of communication between the cofactor peptide and the ligand in the ligand-binding pocket is a crucial and often discussed issue for the nuclear receptors (NRs), but such allosteric signaling pathways are difficult to detect and the transmission mechanism remains elusive. Here, we apply the anisotropic thermal diffusion method to the LXRα with bound coactivator and ligand. We detected a possible communication pathway between the coactivator peptide and the ligand. The signal is transmitted both through the receptor backbone and side chains. A key signaling residue is the first leucine in the cofactor peptide recognition motif LXXLL, which is conserved within the NR cofactors, suggesting a general mechanism for allosteric signaling. Furthermore, we studied the LXR receptor and cofactor molecular interactions in detail using molecular dynamics simulations. The protein-protein interaction patterns in the complexes of nine different cofactor peptides and holo-LXRα were characterized, revealing the importance of the receptor-cofactor charge clamp interaction. Specific, but infrequently occurring interactions were observed toward the cofactor peptide C-terminal residues. Thus, additional specificity between LXRα and its cofactors is likely to be found in molecular interactions outside the cofactor peptide or in other biological factors. Show less

Our goal was to determine whether genetic variation at genes affecting statin metabolism or targets of statin therapy would influence low density lipoprotein (LDL) cholesterol lowering with pravastatin, baseline heart disease, or cardiac endpoints on trial. We examined associations of single nucleotide polymorphisms (SNPs) at the liver X receptor alpha (LXRA, rs12221497), and the solute carrier organic anion transporter (SLCO1B1, rs4149056 and rs2306283) gene loci with these variables. We studied 5411 participants in PROSPER (PROspective Study of Pravastatin in the Elderly at Risk) (mean age 75.3 years), who had been randomized to pravastatin 40 mg/day or placebo and were followed for a mean of 3.2 years. No relationships between genetic variation at the LXRA gene locus with statin induced LDL lowering response or other parameters were noted. Both the SLCO1B1 rs4149056 (valine for alanine at 174) and the rs2306283 (asparagine for aspartic acid at 130) SNPs affect the amino acid sequence of the SLCO1B1 gene product. No effect of the rs2306283 SNP on any of the variables was noted. However the presence of the rs4149056 SNP was associated with significantly less LDL cholesterol lowering response to pravastatin (wildtype, 71.5% of the population, -37.0%; heterozygotes, 25.8% of the population, -36.0%; and homozygotes, 2.7% of the population, -31.8%, p=0.003 at 6 months, and p=0.022 at 12 months). Our data indicate that the presence of the rs4149056 non-synonymous SNP at the SLCO1B1 gene locus can significantly decrease the pravastatin induced LDL cholesterol lowering response. Show less

Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH. This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-β). Heme-induced HO-1 and LXR-β were suppressed by knockdown of ATF-1, and HO-1 and LXR-β were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-β in turn led to induction of other genes central to cholesterol efflux, such as LXR-α and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. These results show that ATF-1 mediates HO-1 induction by heme and drives macrophage adaptation to intraplaque hemorrhage. Our definition of an ATF-1-mediated pathway for linked protection from foam cell formation and oxidant stress may have therapeutic potential. Show less

There is increasing evidence that the retinoic acid receptor-related orphan receptor α (RORα) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORα in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORα attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor α (LXRα). First, RORα and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORα, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRα and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORα interacted physically with LXRα and inhibited the LXRα response element in the promoter of LXRα, indicating that RORα interrupts the autoregulatory activation loop of LXRα. Third, infection with adenovirus encoding RORα suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORα protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORα via tail-vein injection of adenovirus (Ad)-RORα decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORα, thereby inducing activation of AMPK and repression of LXRα. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. We found that RORα induced activation of AMPK and inhibition of the lipogenic function of LXRα, which may be key phenomena that provide the beneficial effects of RORα against hepatic steatosis. Show less

Accumulation of amyloid β-peptide (Aβ) in the brain of Alzheimer's disease (AD) patients has been postulated to reflect defects in Aβ degradation or clearance. Here, we selected 12 genes (MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9) involved in Aβ catabolism on the basis of PubMed-based literature search and elucidated their genetic role in AD among Finnish case-control cohort consisting of total ∼1,300 AD patients and control subjects. Thirty one single nucleotide polymorphisms (SNPs) were selected for genotyping. In a smaller subset of AD patients, cerebrospinal fluid (CSF) levels of Aβ42 (n = 124), total-tau (n = 59), and phospho-tau (n = 54) analyses were performed with respect to SNPs. Moreover, age of onset analyses with respect to the studied SNPs were conducted among the AD patient cohort (n = 642). Association analysis of the liver X receptor α (NR1H3) gene SNPs showed a protective effect for C allele carriers of rs7120118 (OR = 0.70, 95% CI 0.53-0.93), while the total-tau and phospho-tau levels in CSF were decreased in AD patients carrying the C allele. Also, a decrease in the age of onset was observed in AD patients carrying the A allele of rs723744 and the C allele of rs3794884 in transthyretin (TTR) gene. However, after adjusting the p-values for multiple comparisons, these results were not statistically significant, suggesting that genetic variations in MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9 genes do not play major role among the Finnish AD patient cohort. Show less

Liver X receptor (LXR) α and LXR β (NR1H3 and NR1H2) are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and β and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD) markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping. By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment. We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers. Show less

An accumulation of data from in vitro to in vivo model system has established a pivotal role of three crucial ligand activated nuclear receptors RXR, LXR-alpha and VDR for their ability to regulate an array of genes involved in regulation of fundamental cellular processes to patho-physiological situations. Keeping in view RXR as a common heterodimeric partner for LXR-alpha and VDR, the present study was designed to dissect the interrelationship between these three nuclear receptors in peripheral blood mononuclear cellular model. The present study revealed that all the three nuclear receptors displayed auto regulation in response to their specific ligands; Both LXR-alpha and VDR regulated the expression of their heterodimeric partner RXR; and VDR was regulated by LXR-alpha through its ability to modulate SREBP response element present in the promoter region of VDR gene. Based on these findings, the role of these nuclear receptors could be better understood in various nuclear receptor mediated pathological processes. Show less

In recent times, the role of LXRs in skin physiology and pathology has evolved rapidly because of their role in proliferation, carcinogenesis, differentiation and permeability barrier function. LXRs were identified as promising drug targets for the treatment of many skin diseases. For this study, skin biopsies were taken from 15 patients with vitiligo and six controls to culture melanocytes from clinically active perilesional and normal skin. Gene expression was examined by reverse transcriptase-polymerase chain reaction analysis. Role of LXR-α in regulating the expression of MMPs was checked by gene knock-down, and its role in vitiligo pathogenesis was checked by treatment with LXR-α agonist 22(R)-hydroxycholesterol. After treatment adhesion assay, annexin V staining and proliferation assay were performed. The expression of LXR-α was relatively more in perilesional skin melanocytes as compared to uninvolved skin melanocytes of non-segmental vitiligo patient, and controls on the other hand, perilesional melanocytes were more prone to apoptosis. LXR-α gene knock-down significantly increases the expression of MMPs. LXR-α agonist 22(R)-hydroxycholesterol treatment significantly decreases melanocyte adhesion, apoptosis and proliferation. Higher expression of LXR-α in perilesional skin melanocytes significantly decreases the adhesion, proliferation and matrix metalloproteinases and increases apoptosis. Show less

The aim of the present study was to test the hypotheses that exercise is associated with generation of peroxisome proliferator-activated receptor-γ (PPARγ) ligands in the plasma and that this may activate PPARγ signaling within circulating monocytes, thus providing a mechanism to underpin the exercise-induced antiatherogenic benefits observed in previous studies. A cohort of healthy individuals undertook an 8-wk exercise-training program; samples were obtained before (Pre) and after (Post) standardized submaximal exercise bouts (45 min of cycling at 70% of maximal O(2) uptake, determined at baseline) at weeks 0, 4, and 8. Addition of plasma samples to PPARγ response element (PPRE)-luciferase reporter gene assays showed increased PPARγ activity following standardized exercise bouts (Post/Pre = 1.23 ± 0.10 at week 0, P < 0.05), suggesting that PPARγ ligands were generated during exercise. However, increases in PPARγ/PPRE-luciferase activity in response to the same standardized exercise bout were blunted during the training program (Post/Pre = 1.18 ± 0.14 and 1.10 ± 0.10 at weeks 4 and 8, respectively, P > 0.05 for both), suggesting that the relative intensity of the exercise may affect PPARγ ligand generation. In untrained individuals, specific transient increases in monocyte expression of PPARγ-regulated genes were observed within 1.5-3 h of exercise (1.7 ± 0.4, 2.6 ± 0.4, and 1.4 ± 0.1 fold for CD36, liver X receptor-α, and ATP-binding cassette subfamily A member 1, respectively, P < 0.05), with expression returning to basal levels within 24 h. In contrast, by the end of the exercise program, expression at the protein level of PPARγ target genes had undergone sustained increases that were not associated with an individual exercise bout (e.g., week 8 Pre/week 0 Pre = 2.79 ± 0.61 for CD36, P < 0.05). Exercise is known to upregulate PPARγ-controlled genes to induce beneficial effects in skeletal muscle (e.g., mitochondrial biogenesis and aerobic respiration). We suggest that parallel exercise-induced benefits may occur in monocytes, as monocyte PPARγ activation has been linked to beneficial antidiabetic effects (e.g., exercise-induced upregulation of monocytic PPARγ-controlled genes is associated with reverse cholesterol transport and anti-inflammatory effects). Thus, exercise-triggered monocyte PPARγ activation may constitute an additional rationale for prescribing exercise to type 2 diabetes patients. Show less

Lygodipenoids A (1) and B (2), two novel C33 tetracyclic triterpenoids with a new 9,19 : 24,32-dicyclopropane skeleton, were isolated from the whole grass of Lygodium japonicum. Their structures were elucidated by spectroscopic and chemical means. Compounds 1 and 2 were tested in transfected cultured human embryonic kidney 293 HEK293 cells for an agonist assay, and compound 1 was identified as a partial agonist for liver X receptor α. Show less

The liver X receptor alpha (LXRα), a member of the nuclear receptor superfamily, has been shown to regulate the expression of the fatty acid synthase (FAS) gene through direct interaction with the FAS promoter. However, its regulation of gene expression is not completely understood. Histone modifications and chromatin remodeling are closely linked to transcriptional activation of genes. In the present study, we examined the effect of LXRα activation or silencing on histone modifications (i.e., acetylation, methylation, and phosphorylation) across the FAS gene, with the aim to investigate whether LXRα could regulate its target gene expression at the epigenetic level. The addition of LXR agonist T0901317 or ectopic expression of LXRα stimulated the FAS transcription, which was coupled with increased levels of histones H3 and H4 acetylation and H3 phosphorylation and methylation at the LXR response element (LXRE). LXR ligation or overexpression induced distinct histone modification patterns at the distal region 2,272 bp upstream from the transcription start site (TSS) and TSS of the FAS gene. Moreover, RNA interference-mediated downregulation of LXRα impaired the histone acetylation and methylation but not phosphorylation on the FAS gene. In conclusion, we provide evidence that LXRα ligation-mediated transcriptional activation of the FAS gene is associated with LXRα-dependent histone acetylation and methylation rather than phosphorylation on this target gene. Show less

Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells. Show less

Fucosterol, a sterol that is abundant in marine algae, has hypocholesterolemic activity, but the mechanism underlying its effect is not clearly understood. Because data suggest that fucosterol can increase plasma high-density lipoprotein concentrations, we investigated whether it could activate liver X receptors (LXRs), critical transcription factors in reverse cholesterol transport. Fucosterol dose-dependently stimulated the transcriptional activity of both LXR-α and -β in a reporter gene assay, responses that were attenuated by the LXR antagonist As(2)O(3). Fucosterol also activated co-activator recruitment in cell-free time-resolved fluorescence resonance energy transfer analysis. In THP-1-derived macrophages, it induced the transcriptional activation of ABCA1, ABCG1, and ApoE, key genes in reverse cholesterol transport, and thereby significantly increased the efflux of cholesterol. Fucosterol also regulated intestinal NPC1L1 and ABCA1 in Caco-2 cells. Notably, fucosterol did not induce cellular triglyceride accumulation in HepG2 cells, primarily because of its upregulation of Insig-2a, which delays nuclear translocation of SREBP-1c, a key hepatic lipogenic transcription factor. These results suggest that fucosterol is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in multiple cell lines without inducing hepatic triglyceride accumulation. Show less

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15dPGJ(2)), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ(2), since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ(2)-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis. Show less

This review focuses on a number of new data on biology and pathophysiology of the metabolic syndrome (MetS) and the involvement of nuclear receptors that have been presented during the last Endocrine Society meeting, held in Houston in June 2012. Several studies have reported beneficial effects of various orphan nuclear receptors, including SHP (Small Heterodimeric Partner, NR0B2) and LXR (Liver X Receptor, NR1H3 and NR1H2), on various components of MetS. By using an inactivation model of SHP, David Moore has shown that SHP exerts "antidiabetic" effects but associated with hepatic steatosis development. He also showed that DLPC (dilauroyl phosphatidylcholine), an unconventional phospholipid, exhibited anti-diabetic properties through its binding to LRH-1 (Liver Receptor Homolog-1, NR5A2), a molecular partner of SHP. Interestingly, Carolyn Cummins investigated LXR α and β isoforms knock-out mice and provided experimental evidence for the detailed mechanisms involved in the deleterious metabolic effects of glucocorticoids, pointing out to the functional interaction between LXRβ, and the glucocorticoid receptor. These new and original studies open new therapeutic opportunities for the management of metabolic disorders in humans by selective modulators of these receptors. Show less

The expressions of genes involved in cholesterol efflux increase, whereas those involved in extracellular cholesterol uptake decrease, during spontaneous functional regression of the primate corpus luteum (CL). This may result from liver x receptor (LXR) alpha (official symbol NR1H3) and/or beta (official symbol NR1H2) control of luteal gene transcription, because these nuclear receptor superfamily members are key regulators of cellular cholesterol homeostasis. Therefore, studies were conducted to assess endogenous LXR ligands in the primate CL through the luteal phase, and to determine the effect of synthetic or natural LXR ligands on cholesterol efflux and uptake in functional primate luteal cells. Using high-performance liquid chromatography tandem mass spectrometry, three LXR ligands were identified and quantified in the rhesus macaque CL, including 22R-hydroxycholesterol (22ROH), 27-hydroxycholesterol (27OH), and desmosterol. Levels of 22ROH paralleled serum progesterone concentrations, whereas mean levels of 27OH tended to be higher following the loss of progesterone synthesis. Desmosterol was present throughout the luteal phase. Functional macaque luteal cells treated with the synthetic LXR agonist T0901317 or physiologically relevant concentrations of the endogenous luteal ligands 22ROH, 27OH, and desmosterol had increased expression of various known LXR target genes and greater cholesterol efflux. Additionally, T0901317 reduced low-density lipoprotein receptor protein and extracellular low-density lipoprotein uptake, whereas 27OH decreased low-density lipoprotein receptor protein, most likely via a posttranslational mechanism. Collectively, these data support the hypothesis that LXR activation causes increased cholesterol efflux and decreased extracellular cholesterol uptake. In theory, these effects could deplete the primate CL of cholesterol needed for steroidogenesis, ultimately contributing to functional regression. Show less