Isolation and characterization of the circulating truncated form of PCSK9.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.

Liver X receptors (LXRs) regulate apolipoprotein AIV-implications of the antiatherosclerotic effect of LXR agonists.

Liver X receptors (LXRs) regulate target genes that are critical in lipoprotein metabolism and atherosclerosis. Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is associated with chylomicrons and high-density lipoproteins. Plasma ApoAIV level in humans is inversely correlated with coronary artery events and overexpression of ApoAIV in mice results in significant reduction in atherosclerosis. We report here that LXRs directly regulate apoAIV at the transcriptional level. Treatment of C57B6 mice with a synthetic LXR agonist, T0901317, resulted in significant increases in plasma apoAIV that was associated with high-density lipoprotein. Examination of both intestinal and liver apoAIV mRNA revealed specific increases in liver mRNA only. In a human heptoma HepG2 cell model, apoAIV mRNA was up-regulated upon the treatment with either native or synthetic LXR agonists. Nuclear run-on study revealed a significant increase in the ApoAIV transcriptional rate upon LXR activation. Examination of the human apoAIV proximal promoter revealed a potential LXR response element that demonstrated binding with HepG2 nuclear extracts. Cotransfection studies in HepG2 cells indicated that this responsive element was functional in mediating the human ApoAIV gene response to LXR agonists. In addition, we identified a functional LXR-responsive element at 3' end enhancer region of mouse ApoAIV gene. We conclude that ApoAIV is a direct target gene of LXRs that may contribute to the antiatherogenic effect of LXR activation.

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes.

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.

A natural product ligand of the oxysterol receptor, liver X receptor.

Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXRalpha and LXRbeta; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXRalpha and LXRbeta in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.

A chemical switch regulates fibrate specificity for peroxisome proliferator-activated receptor alpha (PPARalpha ) versus liver X receptor.

Fenofibrate is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia presumably through peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent induction of genes that control fatty acid beta-oxidation. Lipid homeostasis and cholesterol metabolism also are regulated by the nuclear oxysterol receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta). Here we show that fenofibrate ester, but not fenofibric acid, functions as an LXR antagonist by directly binding to LXRs. Likewise, ester forms, but not carboxylic acid forms, of other members of the fibrate class of molecules antagonize the LXRs. The fibrate esters display greater affinity for LXRs than the corresponding fibric acids have for PPARalpha. Thus, these two nuclear receptors display a degree of conservation in their recognition of ligands; yet, the acid/ester moiety acts as a chemical switch that determines PPARalpha versus LXR specificity. Consistent with its LXR antagonistic activity, fenofibrate potently represses LXR agonist-induced transcription of hepatic lipogenic genes. Surprisingly, fenofibrate does not repress LXR-induced transcription of various ATP-binding cassette transporters either in liver or in macrophages, suggesting that fenofibrate manifests variable biocharacter in the context of differing gene promoters. These findings provide not only an unexpected mechanism by which fenofibrate inhibits lipogenesis but also the basis for examination of the pharmacology of an LXR ligand in humans.

Liver X receptor and retinoic X receptor mediated ABCA1 regulation and cholesterol efflux in macrophage cells-messenger RNA measured by branched DNA technology.

ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA. By using the bDNA method, we show that both LXR and RXR ligands effectively regulate ABCA1 expression in three macrophage cell types: mouse RAW264.7 cell line, mouse peritoneal macrophage cells, and human macrophage THP-1 cells and their regulation is additive. Furthermore, by using a radiolabeled cholesterol efflux assay, we show that both LXR and RXR ligands are sufficient to mediate cholesterol efflux in macrophage cells and their efficacy correlates with ABCA1 regulation. These studies strengthen further the notion that LXR and RXR mediate ABCA1 expression and cholesterol efflux in macrophage cells as a permissive heterodimer and development of small molecule ligands of these nuclear receptors may represent a promising approach to modulating cholesterol efflux and plasma HDL cholesterol level in humans.

Phospholipid transfer protein is regulated by liver X receptors in vivo.

Liver X receptors (LXR) belong to the nuclear receptor superfamily that can regulate important lipid metabolic pathways. The plasma phospholipid transfer protein (PLTP) is known to mediate transfer of phospholipids from triglyceride-rich lipoproteins to high density lipoprotein (HDL) and plays a critical role in HDL metabolism. We report here that a specific LXR agonist, T0901317, elevated HDL cholesterol and phospholipid in C57/BL6 mice and generated enlarged HDL particles that were enriched in cholesterol, ApoAI, ApoE, and phospholipid. The appearance of these HDL particles upon oral dosing of T0901317 in C57/BL6 mice was closely correlated with the increased plasma PLTP activity and liver PLTP mRNA levels. Nuclear run-on assay indicated that the effect of LXR agonist on PLTP expression was at the transcriptional level. In mouse peritoneal macrophage cells, PLTP expression was also up-regulated by the LXR/RXR (retinoid X receptor) heterodimer. However, cholesterol efflux in mouse peritoneal macrophage cells from PLTP-deficient mice (PLTP0) was not significantly different from wild type animals. Although in PLTP-deficient mice, the induction of HDL cholesterol as well as HDL particle size increase persisted, the extent of the induction was greatly attenuated. We conclude that PLTP is a direct target gene of LXRs in vivo and plays an important role in LXR agonist-mediated HDL cholesterol and size increase in mice.

Estrogen receptor-mediated repression of human hepatic lipase gene transcription.

Estrogen replacement therapy in women decreases hepatic lipase (HL) activity, which may account for the associated increase in HDL cholesterol. To investigate whether estrogen decreases HL transcription, transient cotransfection assays with HL promoter and estrogen receptor-alpha (ERalpha) expression constructs were performed in HepG2 cells. 17beta-estradiol (E(2)) decreased transcription driven by the -1557/+41 human HL promoter by up to 50% at 10(-7) M. Mutation of ERalpha by deletion of its transactivation domains or ligand-binding domain eliminated E(2)-induced repression of the promoter, whereas deletion of the DNA-binding domain of ERalpha resulted in a 7-fold activation by E(2). The E(2)-induced repression was maintained after mutation of a potential estrogen-response element in the promoter. The region of estrogen responsiveness was localized to -1557/-1175 of the HL promoter by deletion analysis. Mutation of an AP-1 site at -1493 resulted in a partial loss of E(2)-induced repression, similar to that caused by deletion of nucleotides -1557 to -1366. Gel shift assays with nuclear extracts from E(2)-treated HepG2 cells stably expressing ERalpha demonstrated an increase in binding to an AP-1 consensus oligonucleotide. The AP-1 activator, phorbol 12-myristate 13-acetate, inhibited the HL promoter by greater than 50%. Collectively, the data suggest that estrogen represses the transcription of the HL gene, possibly through an AP-1 pathway.