Cell surface expression of LDL receptor in chronic hepatitis C: correlation with viral load

The LDL receptor (LDL-R) has been proposed as the viral receptor for Hepatitis C virus (HCV). This hypothesis has been based exclusively on in vitro studies. In human mononuclear cells, LDL-R gene expression has been demonstrated to be parallel and be coordinately regulated to gene expression in the human liver. The purpose of the current study was to determine the mononuclear cell surface expression of the LDL receptor in patients with HCV chronic infection according to viral load. Sixty-eight consecutive untreated chronic hepatitis C patients were studied to determine the mononuclear cell surface expression of the LDL-R. LDL-Rs were quantified at the surface of mononuclear cells in fresh blood samples taken after fasting using flow cytometry. LDL-R expression was significantly associated with LDL-cholesterol (r = -0.25; P = 0.03) and HCV-viral load (r = 0.37, P = 0.002). In multivariate analysis, the LDL-R expression was significantly associated with HCV viral load, whereas genotype, age, body mass index, and fibrosis were not. In conclusion, our data provided by a human study, suggest that the LDL-R may be one of the receptors implicated in HCV replication.     

家族性高胆固醇血症样表型遗传异质性的分子基础

家族性高胆固醇血症(FH)是由于低密度脂蛋白受体(LDL—R)基因突变,致使细胞表面LDL-R蛋白功能缺陷,导致血浆低密度脂蛋白(LDL)大幅度增高,并可导致早发冠心病。“FH”已经成为携带LDL-R基因突变患者的同意词,但日益增多的研究证实,其他6种基因突变也可通过不同机制导致FH样表型。这些致病基因的发现．促进胆固醇代谢的研究进入新领域,有助于深入探讨胆固醇代谢的调节机制,并将为FH样表型的诊断和治疗提供新的理论依据。文章就有关FH样表型遗传异质性的分子基础研究的近况作一简要综述．以引起人们的关注。     

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.     

Hypercholesterolemia and LDL receptor mRNA expression: modulation by selenium supplementation

Selenium (Se) status has been associated with cardiovascular disorders. Present study was aimed to elucidate the protective role of Se supplementation on LDL receptor (LDL-R) activity as well as mRNA expression during experimental hypercholesterolemia in SD male rats. Animals were fed 0.2 and 1 ppm Se supplemented control diet as well as 2% cholesterol supplemented diet for 3 months. LDL-R activity was measured in-vivo by injecting radiolabeled LDL to rats and decrease in counts per minute with time was taken as a measure of LDL clearance and in turn LDL-R activity. LDL-R mRNA expression was studied by RT-PCR. LDL-R activity and mRNA expression decreased significantly on 2% cholesterol supplemented diet feeding. On 1 ppm Se supplementation LDL-R activity as well as mRNA expression increased significantly. Present results demonstrate that Se supplementation upto 1 ppm is responsible for up regulation of LDL-R activity as well as mRNA expression, during hypercholesterolemia. These findings highlight the therapeutic potential of Se supplementation in lipid metabolism.     

Localization of apolipoprotein E receptor 2 to caveolae in the plasma membrane

The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.     

Attenuation of LDL receptor gene expression by selenium deficiency during hypercholesterolemia

Selenium deficiency has been associated with hypercholesterolemia. Present study was aimed to determine the effect of selenium (Se) deficiency on LDL receptor (LDL-R) activity as well as mRNA expression during experimental hypercholesterolemia in SD male rats. Animals were fed Se adequate (0.2 ppm) and deficient (0.02 ppm) control diet as well as high cholesterol (2%) diet (HCD) for 1 and 2 months. LDL-R activity was measured in vivo by injecting radiolabeled LDL to rats and percent decrease in cpm with time was taken as a measure of LDL clearance and in turn LDL-R activity. LDL-R mRNA expression was studied by RT-PCR. LDL-R activity and mRNA expression decreased significantly on HCD feeding in both Se deficient and adequate diet fed rats after 2 months. In Se deficiency receptor activity and mRNA expression decreased significantly. After 2 months LDL-R activity and expression decreased in both the Se deficient groups and in Se adequate HCD fed group in comparison to 1 month data. But after 4 month there was no significant difference observed in LDL-R activity and mRNA expression in selenium deficiency as well as on HCD feeding. So the present results demonstrate that Se deficiency act synergistically with hypercholesterolemia to downregulate LDL-R activity as well as mRNA expression.     

In vivo regulation of hepatic LDL receptor mRNA in the baboon. Differential effects of saturated and unsaturated fat

The effects of diets enriched with cholesterol and different fats upon plasma lipoproteins and hepatic low density lipoprotein (LDL) receptor mRNA levels were studied in a group of 18 normal baboons. Animals were fed diets containing 1% cholesterol and 25% fat as either coconut oil, peanut oil, or olive oil for a period of 20 weeks. Plasma total cholesterol, high density lipoprotein (HDL) cholesterol, beta-lipoprotein (LDL + very low density lipoprotein) cholesterol, apolipoprotein B and apolipoprotein A-I were measured in samples obtained at 4-week intervals. All three diet groups demonstrated a statistically significant increase in plasma cholesterol as compared to base line throughout the experiment. Hepatic LDL receptor (LDL-R) mRNA levels were quantified by dot blot hybridization in serial liver biopsies. Animals fed saturated fat sustained a significant reduction in hepatic LDL-R mRNA as compared to those fed either monounsaturated or polyunsaturated fat. A strong negative correlation between LDL-R mRNA and plasma total cholesterol (r = -0.71), HDL cholesterol (r = -0.76), and plasma apo A-I (r = -0.77) was observed only in those animals fed coconut oil. Weak negative correlations between LDL-R mRNA and other plasma parameters did not achieve statistical significance. We conclude that saturated and unsaturated oils may influence plasma cholesterol levels in part through differential effects on LDL receptor biosynthesis in baboons.     

The effect of heparin on structural and functional properties of low density lipoproteins

Heparin binding to human low density lipoproteins (LDL) and the effect of heparin on the ability of LDL to bind to the LDL receptor has been investigated. Emphasis has been made on the physiological conditions of temperature, pH and the ionic strength. Intrinsic fluorescence spectroscopy of LDL has been applied to follow heparin binding. Fluorescence anisotropy has been measured to describe the changes in apoB and dansyl-heparin dynamics upon binding. Eu3+-labeled LDL binding to the intact LDL receptor has been monitored by time-resolved fluorescence spectroscopy technique. We have found that heparin binds to LDL under the physiological conditions, probably by Van der Waals interactions and hydrogen bonding. Temperature seems to be the most important factor influencing the interaction. Furthermore, the presence of heparin inhibits LDL binding to the intact LDL receptor that might have consequences on the cholesterol metabolism in vivo.     

Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers

Proteins of the low-density lipoprotein receptor family transport cholesterol-carrying particles into cells, clear protease-inhibitor complexes from the circulation, participate in biological signaling cascades, and even serve as viral receptors. These receptors utilize clusters of cysteine-rich LDL receptor type-A (LA) modules to bind many of their ligands. Recent structures show that these modules typically exhibit a characteristic binding mode to recognize their partners, relying primarily on electrostatic complementarity and avidity effects. The dominant contribution of electrostatic interactions with small interface areas in these complexes allows binding to be regulated by changes in pH via at least two distinct mechanisms. The structure of the subtilisin/kexin family protease PCSK9, a newly identified molecular partner of the LDLR also implicated in LDL-cholesterol homeostasis, also raises the possibility that the LDLR and its related family members may employ other strategies for pH-sensitive binding that have yet to be uncovered.     

Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: a flow cytometric analysis

Dormant lymphocytes are known to show little LDL receptor (LDL-R) activities. The present study was designed to determine whether or not LDL-R activities of lymphocytes from normal subjects were high enough to be measured by flow cytometry after the cells had been stimulated with recombinant interleukin-2 (IL-2) and anti-CD3 monoclonal antibody (mAb). IL-2 or anti-CD3 mAb individually provokes proliferation of lymphocytes in a serum-free medium. Proliferation rate was accelerated when the two reagents were used in combination. Stimulated cells cultured for 5 days expressed more than 85% CD3 positive, less than 0.5% CD14 positive, and less than 1.5% CD20 positive. The LDL-R activities of the cells were examined by the uptake of a fluorescence probe, DiI-labeled LDL (DiI-LDL) and analyzed by flow cytometry. Stimulated cells showed increased uptake of DiI-LDL and 84 +/- 9% were positive, whereas only 3.0 +/- 2.5% of the cells without stimulation were positive (P less than 0.001). Under the same conditions stimulated lymphocytes from a homozygous familial hypercholesterolemia (FH) patient showed little LDL-R activities; 14% of the cells were positive. Displacement assays reveal that the uptake of LDL by these cells is occurring by way of its specific pathway. These data imply the lymphocytes stimulated with the reagents used in the study might be used for detecting defects in LDL-R, perhaps defects in other genomic systems as well.     

Intrauterine growth restriction is associated with alterations in placental lipoprotein receptors and maternal lipoprotein composition

Among other factors, fetal growth requires maternal supply of cholesterol. Cellular cholesterol uptake is mainly mediated by the LDL receptor (LDL-R) and the scavenger receptor family. We hypothesized that expression levels of key receptors of these families were regulated differently in placentas from IUGR pregnancies with varying degrees of severity. Third-trimester placentas from IUGR pregnancies with (IUGR-S) and without (IUGR-M) fetal hemodynamic changes and from control (AGA) pregnancies were studied. LDL-R, LDL-R-related protein (LRP-1), and scavenger receptor class B type I (SR-BI) mRNA and protein levels were measured. Cholesterol concentration and composition of lipoproteins were analyzed enzymatically and by lipid electrophoresis, respectively, in maternal and umbilical cord blood. LDL-R mRNA levels in IUGR-M were similar to AGA but lower (P < 0.05) in IUGR-S. In contrast, LDL-R protein was twofold (IUGR-M) and 1.8-fold (IUGR-S) higher (P < 0.05) than in the AGA group. LRP-1 mRNA and protein levels were not altered in the IUGR cases. SR-BI mRNA was unchanged in IUGR, but protein levels were lower (P < 0.05) in IUGR-S than in the other groups. Maternal plasma concentrations of LDL cholesterol were higher (P < 0.05) in the AGA group (188.5 +/- 23.6 mg/dl) than in the IUGR-S group (154.2 +/- 26.1). Electrophoretic mobility of the LDL fraction in maternal plasma demonstrated significant changes in migration toward higher values (AGA 0.95 +/- 0.06, IUGR-M 1.12 +/- 0.11, P < 0.001; IUGR-S 1.28 +/- 0.20, P = 0.002). We conclude that LDL-R and SR-BI levels are altered in IUGR pregnancies. These differences were associated with changes in LDL, but not HDL, mobility and cholesterol concentration in maternal circulation.     

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.     

Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor

Low-density lipoprotein (LDL) receptors bind lipoprotein particles at the cell surface and release them in the low pH environment of the endosome. The published structure of the receptor determined at endosomal pH reveals an interdomain interface between its beta propeller and its fourth and fifth ligand binding (LA) repeats, suggesting that the receptor adopts a closed conformation at low pH to release LDL. Here, we combine lipoprotein binding and release assays with NMR spectroscopy to examine structural features of the receptor promoting release of LDL at low pH. These studies lead to a model in which the receptor uses a pH-invariant scaffold as an anchor to restrict conformational search space, combining it with flexible linkers between ligand binding repeats to interconvert between open and closed conformations. This finely tuned balance between interdomain rigidity and flexibility is likely to represent a shared structural feature in proteins of the LDL receptor family.     

Calmodulin antagonists increase the amount of mRNA for the low-density-lipoprotein receptor in skin fibroblasts.

The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a 'Northern' blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA.     

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.     

Mathematical Model for Low Density Lipoprotein (LDL) Endocytosis by Hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL–LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.     

Chondroitin sulfate-modified LDL induces increased cholesteryl ester synthesis and down-regulation of LDL receptors in smooth muscle cells and macrophages

Hypercholesterolemia induces increased transcytosis and accumulation of plasma lipoproteins in the arterial intima, where they interact with matrix proteins and become modified and reassembled lipoproteins. Chondroitin 6-sulfate-modified LDL (CS-mLDL) induces migration, proliferation, and lipid accumulation in human aortic smooth muscle cells (SMCs). To search for the mechanism(s) responsible for lipid accumulation, cultured SMC and macrophages were exposed to CS-mLDL, minimally modified LDL (mmLDL), and native LDL (as a control). Then the cellular uptake, degradation and expression of the LDL receptor (LDL-R) was determined using radioiodinated ligands, ACAT activity assay, fluorescence microscopy and RT-PCR. The uptake of CS-mLDL was 2-fold higher in SMC and 3-to 4-fold higher in macrophages as compared to LDL and mmLDL; the lysosomal degradation of CS-mLDL was slower in SMCs and considerably diminished in macrophages. Compared with LDL, CS-mLDL induced increased synthesis and accumulation of esterified cholesterol in SMCs (∼2-fold) and macrophages (∼10-fold) within an expanded acidic compartment. CS-mLDL and mmLDL down-regulate the gene expression of the LDL-R in the both cell types. Mechanisms of CS-mLDL-induced lipid accumulation in SMC and macrophages involve increased cellular uptake, and diminished cellular degradation that stimulates cholesterol ester synthesis and accumulation in cytoplasmic inclusions and in the lysosomal compartment in an undegraded form; modified lipoproteins induce down-regulation of LDL-R.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line ¹²⁵I-LDL, and on a murine macrophage cell line (J774) ¹²⁵I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.