t0901317 and 25-hydroxycholesterol

The pathogenesis of chronic obstructive pulmonary disease (COPD) is associated with dyslipidemia, an established co-morbidity. Statins treat hypercholesterolemia, but more recently have been trailed in the setting of COPD for their potential anti-inflammatory benefits. The outcomes of prospective trials however have been inconsistent. Thus, we hypothesize that the variation in results may have been due to statin-induced downregulation of ATP-binding cassette transporter A1 (ABCA1), thereby reducing cholesterol export. This study aims to elucidate whether statin treatment in a cellular model of COPD leads to a decrease in ABCA1 protein expression.. To mimic the inflammatory environment of COPD, two commonly used lung epithelial cell lines (BEAS-2B and A549) were treated with tumor necrosis factor (TNF), and co-treated with cholesterol/25-hydroxycholesterol (25-OH) to mimic dyslipidemia. ABCA1 protein was detected by Western Blotting.. We unexpectedly showed that statins did not affect ABCA1 expression. However, the LXR agonist T0901317 significantly increased ABCA1 expression in both cell lines, while TNF, cholesterol or 25-OH induced ABCA1 protein upregulation in BEAS-2B cells, indicating cell line differences in response. There was also evidence of synergistic impacts of combined treatments on ABCA1 upregulation in BEAS-2B cells.. Statins did not have an impact on ABCA1 expression in lung epithelial cell lines, disproving our original hypothesis. However, we showed for the first time, the effect of the inflammatory cytokine TNF, cholesterol/25-OH, statins and the LXR agonist T0901317 on expression of ABCA1 transporter protein in human lung epithelial cell lines in vitro. We hope that these in vitro studies may prove beneficial for addressing dyslipidemia in COPD in the future.

Topics: A549 Cells; ATP Binding Cassette Transporter 1; Cell Line; Cell Line, Tumor; Cholesterol; Epithelial Cells; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Liver X Receptors; Lung; Sulfonamides; Tumor Necrosis Factor-alpha; Up-Regulation

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and β are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/β in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Topics: Animals; Cell Differentiation; Cerebellum; Cholesterol; Gene Expression Regulation; Homeostasis; Hydrocarbons, Fluorinated; Hydroxycholesterols; Liver X Receptors; Male; Mice; Mice, Knockout; Myelin Sheath; Oligodendroglia; Organ Culture Techniques; Orphan Nuclear Receptors; Promoter Regions, Genetic; Psychomotor Performance; RNA, Messenger; Spatial Learning; Sulfonamides

The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized.. We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts.. Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC.. These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands.

Topics: Cell Differentiation; Cells, Cultured; Cholesterol; Female; Fetal Growth Retardation; Gene Expression; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Ligands; Lipid Droplets; Liver X Receptors; Orphan Nuclear Receptors; Oxidation-Reduction; Pregnancy; Progesterone; Sterol Regulatory Element Binding Protein 2; Sulfonamides; Trophoblasts

ABCA7 is a member of the ABCA subfamily that shows a high degree of homology to ABCA1 and, like ABCA1, mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 has been shown to be downregulated by increased cellular cholesterol while ABCA1 was upregulated.. The underlying mechanism for this effect was examined in ABCA1 or ABCA7-transfected HEC293. Lipid content in the medium and cells was determined by enzymatic assays. Gene expression was quantitated by real time PCR, and protein content was determined by Western blotting.. While ABCA7 mRNA was decreased by 25-hydroxycholesterol treatment, ABCA1 was apparently increased. Treatment with the synthetic LXR agonist T0901317 (T09) upregulated ABCA1 expression and apoAI-mediated cellular lipid release in ABCA1-transfected HEC293 cells, but ABCA7 expression and cellular lipid release in ABCA7-transfected HEC293 cells showed no obvious changes.. The ABCA7 gene is regulated by sterol in a direction opposite to that of ABCA1.

Topics: Amino Acid Sequence; Apolipoprotein A-I; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Gene Expression Regulation; HEK293 Cells; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Lipid Metabolism; Liver X Receptors; Molecular Sequence Data; Orphan Nuclear Receptors; Sulfonamides

25-Hydroxycholesterol (25HC) and its sulfated metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), regulate certain aspects of lipid metabolism in opposite ways. Hence, the enzyme for the biosynthesis of 25HC3S, oxysterol sulfotransferase (SULT2B1b), may play a crucial role in regulating lipid metabolism. We evaluate the effect of 25HC sulfation on lipid metabolism by overexpressing the gene encoding SULT2B1b in human aortic endothelial cells (HAECs) in culture.. The human SULT2B1b gene was successfully overexpressed in HAECs following infection using a recombinant adenovirus. HPLC analysis demonstrated that more than 50% of (3)H-25HC was sulfated in 24h following overexpression of the SULT2B1b gene. In the presence of 25HC, SULT2B1b overexpression significantly decreased mRNA and protein levels of LXR, ABCA1, SREBP-1c, ACC-1, and FAS, which are key regulators of lipid biosynthesis and transport; and subsequently reduced cellular lipid levels. Overexpression of the gene encoding SULT2B1b gave similar results as adding exogenous 25HC3S. However, in the absence of 25HC or in the presence of T0901317, synthetic liver oxysterol receptor (LXR) agonist, SULT2B1b overexpression had no effect on the regulation of key genes involved in lipid metabolism.. Our data indicate that sulfation of 25HC by SULT2B1b plays an important role in the maintenance of intracellular lipid homeostasis via the LXR/SREBP-1c signaling pathway in HAECs.

Topics: Cells, Cultured; Cholesterol Esters; Chromatography, High Pressure Liquid; Endothelial Cells; Fatty Acids, Nonesterified; Gene Expression Regulation; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Liver X Receptors; Orphan Nuclear Receptors; RNA, Messenger; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Sulfonamides; Sulfotransferases; Time Factors; Transfection; Triglycerides

Oxygenated cholesterol metabolites known as oxysterols display potent biological activities ranging from regulation of lipid homeostasis to cytotoxicity. Oxysterols have previously been shown to inhibit the invasion of first trimester trophoblasts, an effect which involves activation of the nuclear liver X receptors (LXRs). In the present study, we investigated the effects of several oxysterols on syncytialisation (differentiation and fusion) in term placental trophoblasts. Treatment of cultured term primary trophoblast cells with oxysterols [25-hydroxycholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol] and the synthetic LXR agonist T0901317 at non-toxic doses decreased expression of GCM-1 and HERV-W mRNA and reduced hCG secretion and placental alkaline phosphatase activity, indicative of diminished trophoblast differentiation. Furthermore, treatment with these compounds also decreased cell fusion measured by E-cadherin immunostaining and quantification of syncytialised nuclei. Treatment with an LXR antagonist (geranylgeranyl diphosphate) abrogated the inhibitory effects of oxysterols and T0901317 on trophoblast syncytialisation indicating that these effects are mediated by LXR. These findings suggest that oxysterols impair differentiation and fusion of term trophoblast cells via an LXR-dependent mechanism.

Topics: Alkaline Phosphatase; Cell Differentiation; Cell Fusion; Chorionic Gonadotropin; DNA-Binding Proteins; Female; Gene Products, env; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Ketocholesterols; Liver X Receptors; Nuclear Proteins; Orphan Nuclear Receptors; Placenta; Pregnancy; Pregnancy Proteins; RNA, Messenger; Sulfonamides; Transcription Factors; Trophoblasts

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.

Topics: Animals; Apoptosis; DNA-Binding Proteins; Enzyme Activation; Flow Cytometry; Gas Chromatography-Mass Spectrometry; Group II Phospholipases A2; Hydrocarbons, Fluorinated; Hydroxycholesterols; Liver X Receptors; Mice; Microscopy, Atomic Force; Oligodendroglia; Orphan Nuclear Receptors; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Transfection

Recent evidence suggests that the liver X receptor (LXR) is a potential anticancer target in prostate carcinoma. There is little characterization, however, of which of the two LXR isoforms, LXRalpha or LXRbeta, regulates the LXR-responsive genes ATP-binding cassette subfamily members A1 (ABCA1) and G1 (ABCG1) in transformed prostatic epithelial cells. In this study, small interfering RNA (siRNA) was used to determine whether LXRalpha or LXRbeta is involved in regulating ABCA1 and ABCG1 mRNA expression in LNCaP and PC-3 cells. Treatment of both cell lines with the synthetic LXR ligand T0901317 and oxysterols: 25-hydroxycholesterol (25HC) and 24(S), 25-epoxycholesterol (24,25EC), resulted in more than a 10-fold increase of ABCA1 and ABCG1 mRNA expression. Transfection of LNCaP cells with siRNA against either LXRbeta or LXRalpha failed to inhibit T0901317 and 25HC-mediated increase of ABCA1 mRNA. siRNA silencing of LXRbeta did, however, inhibit ABCA1 mRNA expression in 24,25EC-treated LNCaP cells. In contrast, LXRbeta siRNA inhibited T0901317, 25HC, and 24,25EC induction of ABCA1 mRNA in PC-3 cells and ABCG1 mRNA in both LNCaP and PC-3 cells. Additional experiments revealed that T0901317 and 25HC induction of ABCA1 mRNA expression was significantly inhibited by the p38 stress kinase antagonist SB202190 and PKA inhibitor H89. Our study is the first to show that LXRbeta, but not LXRalpha, is the major regulatory isoform of ABCG1 mRNA expression in LNCaP and PC-3 cells. Our study also reveals that ABCA1 gene expression is differentially regulated by synthetic and natural LXR ligands, possibly involving kinase mediated signal transduction.

Topics: ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Cholesterol; DNA-Binding Proteins; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Ligands; Liver X Receptors; Male; Neoplasms, Hormone-Dependent; Orphan Nuclear Receptors; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Tumor Cells, Cultured

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRalpha knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Gene Expression Regulation; Hep G2 Cells; Hepatocytes; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Immunoenzyme Techniques; Liver; Liver Neoplasms; Liver X Receptors; Male; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Orphan Nuclear Receptors; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sulfonamides; Transfection

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.

Topics: Androstenes; Animals; Benzoates; Benzylamines; Carbazoles; Cell Line, Tumor; Cholesterol; Ciona intestinalis; DNA-Binding Proteins; Evolution, Molecular; Humans; Hydrocarbons, Fluorinated; Hydroxycholesterols; Liver X Receptors; Mice; Molecular Structure; Orphan Nuclear Receptors; Phylogeny; Receptors, Cytoplasmic and Nuclear; Structure-Activity Relationship; Sulfonamides; Xenopus laevis; Zebrafish