Chinese hamster ovary cell mutant with defective down-regulation of low density lipoprotein receptors

A monensin-resistant clone (Monr-31) shows a related series of differences from its parental Chinese hamster ovary (CHO) cell line in the cellular response to several ligands. The uptake and metabolism of low density lipoprotein (LDL) in the mutant cells are defective. Accumulation of fluorescent-labeled LDL as well as internalization and degradation of 125I-LDL are greatly reduced in Monr-31 cells. The receptor number for LDL on the cell surface of Monr-31 is about one-third that for CHO cells, but affinity constants for both cell lines are similar. Electrophoretic analysis shows a slightly reduced molecular weight of LDL receptor in Monr-31 cells in comparison to that in CHO cells. The internalization index (internalization plus degradation per binding) of LDL of the mutant is about one-half that of CHO cells, suggesting a failure of internalization of LDL as well as LDL binding. Hybrids (hyb-1, -2, and -3) between CHO and Monr-31 cells show LDL binding and LDL internalization activities comparable to that of CHO cells, suggesting that the altered LDL response in Monr-31 cells is recessive. Addition of exogenous LDL to culture medium down-regulates the LDL receptor activity of CHO, hyb-2, and hyb-3 cells, whereas no such down-regulation is seen in Monr-31 cells. Probably as a result of the failure of down-regulation, the prominent inhibition of sterol synthesis from acetate and 3-hydroxy-3-methylglutaryl-coenzyme A reductase observed in CHO cells is scarcely detectable in Monr-31 cells. As a correlated result, sterol synthesis from acetate is 6-fold higher in the mutant. The failure of down-regulation of LDL receptors in Monr-31 cells is discussed in relation to the altered binding and internalization of LDL.     

Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein

This paper describes a procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of the unique structure of low density lipoprotein, a plasma cholesterol transport protein that enters cells by receptor-mediated endocytosis. LDL contains a core of cholesteryl ester that can be extracted and reconstituted with hydrophobic molecules that convert the LDL into a toxic or fluorescent particle. Mutagenized Chinese hamster ovary cells were incubated with reconstituted LDL containing toxic 25-hydroxycholesteryl oleate. Wild-type cells take up this lipoprotein via the LDL receptor, liberate the 25-hydroxycholesterol in lysosomes, and die. To identify colonies of receptor-deficient cells from among the few survivors of the first selection step, we incubated the cells with LDL reconstituted with a fluorescent cholesteryl ester and picked colonies that failed to accumulate fluorescence. The two-step isolation procedure yielded receptor-deficient cells at a frequency of 1 in 10⁵. The mutant cells grew in the presence of LDL reconstituted with 25-hydroxycholesteryl oleate at concentrations 100-fold higher than those that killed parental cells. The altered phenotypes have remained stable for more than 200 population doublings under non-selective conditions. Inasmuch as LDL can be coupled to ligands that bind to receptors other than the LDL receptor, the above method may have general utility in isolating cells with mutations affecting other receptor systems.     

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.     

Superinduction of the human gene encoding low density lipoprotein receptor

Expression of human LDL receptor mRNA and protein is induced in human glioblastoma-astrocytoma cells upon transfer into lipoprotein-deficient medium, a mode of induction likely to be physiological. The presence of cycloheximide (CHX) leads to up to 7.5-fold superinduction of LDL receptor mRNA within 4 hr and, upon removal of this inhibitor, to superinduction of LDL receptor protein as well. The extent of superinduction of LDL receptor mRNA reaches over 40-fold beyond the level expressed in medium containing regular serum. Despite its extensive superinduction, LDL receptor mRNA decays rapidly in the presence of CHX. Stabilization of LDL receptor mRNA is thus unlikely to account for the observed superinduction. These results show that normally the LDL receptor gene is expressed to only a small fraction of its full potential.     

Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family

Apolipoprotein A-V is a potent modulator of plasma triacylglycerol levels. To investigate the molecular basis for this phenomenon we explored the ability of apolipoprotein A-V, in most experiments complexed to disks of dimyristoylphosphatidylcholine, to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA. Experiments using surface plasmon resonance showed specific binding of both free and lipid-bound apolipoprotein A-V to both receptors. The binding was calcium dependent and was inhibited by the receptor associated protein, a known ligand for members of the low density lipoprotein receptor family. Preincubation with heparin decreased the receptor binding of apolipoprotein A-V, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double mutant, apolipoprotein A-V (Arg210Glu/Lys211Gln), showed decreased binding to heparin and decreased ability to bind the low density lipoprotein receptor-related protein. Association of apolipoprotein A-V with the low density lipoprotein receptor-related protein or SorLA resulted in enhanced binding of human chylomicrons to receptor-covered sensor chips. Our results indicate that apolipoprotein A-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins.     

Control of low density lipoprotein receptor gene expression in steroidogenic cells

Low density lipoprotein (LDL)-carried cholesterol is a primary substrate for steroid hormone synthesis by luteinized human granulosa cells. Chorionic gonadotropin and 8-bromo-cAMP both increase LDL receptor levels in granulosa cells by stimulating accumulation of the receptor mRNA. LDL and 25-hydroxycholesterol reduce LDL receptor expression, but this suppressive effect is partially overcome by 8-bromo-cAMP. Using fusion gene constructs containing the LDL receptor gene promoter transfected into JEG-3 cells, a cyclic AMP responsive enhancer could not be identified in the LDL receptor gene upstream promoter in transfection studies. We suggest that the LDL receptor gene in human steroidogenic cells is under negative control by a sterol effector, but that a cyclic AMP triggered process overcomes, to some extent, the sterol-mediated suppression. The detailed mechanisms by which sterol and cyclic AMP modulate LDL receptor gene expression remain to be elucidated.     

Improving lipoprotein profiles by liver-directed gene transfer of low density lipoprotein receptor gene in hypercholesterolaemia mice

The defect of low density lipoprotein receptor disturbs cholesterol metabolism and causes familial hypercholesterolaemia (FH). In this study, we directly delivered exogenous Ldlr gene into the liver of FH model mice (Ldlr^?/?) by lentiviral gene transfer system. The results showed that the Ldlr gene controlled by hepatocyte-specific human thyroxine-binding globulin (TBG) promoter successfully and exclusively expressed in livers. We found that, although, the content of high density lipoprotein in serum was not significantly affected by the Ldlr gene expression, the serum low density lipoprotein level was reduced by 46%, associated with a 30% and 28% decrease in triglyceride and total cholesterol, respectively, compared to uninjected Ldlr^?/? mice. Moreover, the TBG directed expression of Ldlr significantly decreased the lipid accumulation in liver and reduced plaque burden in aorta (32%). Our results indicated that the hepatocyte-specific expression of Ldlr gene strikingly lowered serum lipid levels and resulted in amelioration of hypercholesterolaemia.     

Reconstitution of the low density lipoprotein receptor

The receptor for low density lipoprotein was purified from bovine adrenal cortex in the presence of the nonionic detergent octylglucoside. Receptors were incorporated into the bilayer of egg phosphatidylcholine vesicles by a detergent-dialysis method. Reconstituted receptors were functional in that they bound low density lipoprotein as well as a monoclonal antibody directed against the receptor in a specific, saturable fashion. Binding activity of reconstituted receptors was measured by a gel chromatography assay. The orientation of the receptor molecule within the phospholipid bilayer was investigated by binding assays following proteolytic digestion. Reconstituted receptors showed an orientation that was functionally indistinguishable from that of low density lipoprotein receptors in the plasma membrane of intact human fibroblasts.     

Expression of low density lipoprotein receptor gene in human placenta during pregnancy

Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed.     

Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of human monocytic leukemia cell line (THP-1 cell)

THP-1 cells, a human cell line established from acute monocytic leukemia cells, degraded native human low density lipoprotein (LDL) through a LDL-specific pathway, but had no ability to degrade acetylated LDL. When the cells were treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) to differentiate into the macrophage-like stage, those acquired the ability to degrade acetylated LDL through its specific pathway and lost the ability to degrade native LDL. Degradation of acetylated LDL by the differentiated cells was not reduced by preincubation with either acetylated LDL or native LDL.     

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.     

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Enzyme immunoassay of the receptors for modified low density lipoprotein

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 micrograms/ml. This effect was completely blocked by polyinosinic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.