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.     

Glucocorticoid-related impairment in the metabolism of low density lipoprotein by human fibroblasts

The metabolism of radiolabelled 125I-low density lipoprotein (LDL) was studied in cultured human dermal fibroblasts to investigate potential mechanisms contributing to the accelerated development of cardiovascular disease in patients treated chronically with corticosteroids. Fibroblasts exposed for 48 hours to pooled lipoprotein-poor (d greater than 1.25 gm/ml) serum from glucocorticoid-treated patients showed an increased capacity to bind LDL (p less than .001) compared to cells incubated under identical conditions with pooled serum from controls. In addition, a significantly (p less than .001) reduced amount of soluble radioactive material appeared in the media indicating that exposure of fibroblasts to corticosteroid serum also impaired their capacity to degrade LDL. If this tendency of cultured cells to accumulate cholesterol-rich lipoprotein when exposed to constituents of serum from these patients is present in patients receiving long-term treatment with glucocorticoids, it might influence arterial lipid accumulation and accelerate their developing cardiovascular disease.     

Early appearance of dispersed low density lipoprotein receptors on the fibroblast surface during recycling

We have examined the distributions of recycling low density lipoprotein receptors (LDL-Rs) as they emerge onto and cluster on the surfaces of cultured cells. Surface LDL-Rs were labeled with colloidal gold-LDL conjugates (AuLDL) and cells viewed as whole-mounts in the transmission electron microscope. The steady-state distribution of LDL-Rs on the cell surface, labeled with AuLDL at 4 degrees C, comprised ring-shaped clusters of receptors with dispersed receptors scattered amongst them. After 12 min of incubation at 37 degrees C, virtually all AuLDL probes were internalized. Electron microscopy of thin sections revealed clustered receptors in coated pits and the progressive accumulation of AuLDL in endosomes, multivesicular bodies and lysosomes. By initially blocking all surface LDL-Rs, either with unconjugated LDL or AuLDL of one size, the clustering behavior of newly emerged receptors which recycled to the cell surface was selectively visualized with an AuLDL probe of a second size over a defined time-course. Release of the blocking ligand during the time-course was found to be negligible. Newly appearing dispersed LDL-Rs were detected as early as 2 min and these were often concentrated at the cell margins. The newly labeled and preblocked LDL-Rs did not cocluster before 6 min. By 8 to 12 min, ring-shaped clusters of newly emerged receptors had formed and these were often seen associated with pre-blocked LDL-Rs. The clustering of LDL-Rs on the cell surface was independent of the presence of ligand, AuLDL. Our results indicate that LDL-Rs recycle to the cell surface where they form a dispersed population which gives rise to the ring-shaped clusters of cell surface LDL-Rs associated with coated pits.     

Release of low density lipoprotein receptors from human fibroblasts or HeLa cells by tryptic digestion.

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with $\text{6-[}{}^3\text{H]-glucosamine·HCl}$ and [${}^3\text{H}$]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.     

Effects of phenothiazines on low density lipoprotein metabolism in cultured human fibroblasts

Treatment of cultured human fibroblasts with trifluoperazine or chlorpromazine resulted in a biphasic effect on low density lipoprotein (LDL) catabolism, depending upon the dose. At up to 10^?5 M, a marked increase in LDL binding, internalization and degradation was observed. This phenomenon took place within the first hours of incubation with the drugs, suggesting a direct effect on cell membrane physical characteristics, probably related to the lipophilic properties of phenothiazines. Concentrations above 2 × 10^?5 M resulted in a relative decrease in LDL binding and internalization, and in a dramatic decrease in LDL degradation, which may be related to an inhibition of calmodulin-dependent processes.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia

This paper describes a sensitive method for study of the isoelectric point and molecular weight of immunoreactive low density lipoprotein (LDL) receptors of cultured human fibroblasts. The fibroblast receptors are solubilized with Triton X-100, partially purified by batch elution from DEAE-cellulose, and subjected to two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are transferred electrophoretically to nitrocellulose paper which is then incubated with a mouse monoclonal antibody (IgG-C7) directed against the LDL receptor, followed by an 125I-labeled antibody against mouse IgG. The receptor-bound monoclonal antibody is localized by autoradiography. By this technique, the immunodetectable LDL receptors from normal human fibroblasts migrate as a single spot with an isoelectric point of 4.3 and a Mr of approximately 160,000. In one patient with homozygous familial hypercholesterolemia whose cells fail to bind 125I-labeled IgG-C7, no immunoreactive LDL receptor spot was detected after electrophoresis. We also studied LDL receptors from three homozygotes whose cells bind 125I-IgG-C7, i.e. cross-reacting material-positive mutants. Their immunodetectable receptors were indistinguishable from normal receptors in terms of isoelectric point and molecular weight. Similarly, the receptors from one patient with the internalization-defective form of familial hypercholesterolemia showed normal electrophoretic migration. The immunoblotting technique should prove useful in analyzing structural alterations, if they exist, in LDL receptors from other subjects with cross-reacting material-positive forms of familial hypercholesterolemia.     

Phorbol esters inhibit low density lipoprotein processing by cultured human fibroblasts

A 24 h pretreatment of MRC5 fibroblasts with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a marked decrease in low density lipoprotein (LDL) internalization and degradation; the maximal effect (about 55% decrease) was observed for 10(-7) M TPA. LDL binding was reduced about 35-40%. A significant decrease (about 25%) in LDL internalization was observed after a 2 h incubation of cells with the drug, but longer incubation times (4-6 h) led to a greater effect. Another tumor promoter, phorbol 12,13-dibutyrate decreased LDL internalization by about 35%, whereas the non-tumor promoting 4 alpha-phorbol 12,13-didecanoate had no effect. The protein kinase C inhibitor alpha-cobrotoxin partially antagonized the inhibitory effect of TPA on LDL internalization. The non-phorbol tumor promoter mezerein, another protein kinase C activator, decreased LDL uptake by about 50%. Finally, it was found that TPA had no significant effect on the affinity of the receptor for the LDL. These results suggest a role for protein kinase C in the LDL pathway in cultured human fibroblasts.     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts.

An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals.     

Interaction of swine lipoproteins with the low density lipoprotein receptor in human fibroblasts.

HDLc, a cholesterol-rich lipoprotein that accumulates in the plasma of cholesterol-fed swine, was shown to resemble functionally human and swine low density lipoprotein in its ability to bind to the low density lipoprotein receptor in monolayers of cultured human fibroblasts. This binding occurred even though HDLc lacked detectable apoprotein B, which is the major protein of low density lipoprotein. After it was bound to the low density lipoprotein receptor, HDLc, like human and swine low density lipoprotein, delivered its cholesterol to the cells, and this, in turn, caused a suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, an activation of the cholesterol-esterifying system, and a net accumulation of free and esterified cholesterol within the cells. Swine HDLc, like human high density lipoprotein, did not bind to the low density lipoprotein receptor nor did it elicit any of the subsequent metabolic events. HDLc, like human low density lipoprotein, was incapable of producing a metabolic effect in fibroblasts derived from a subject with the homozygous form of familial hypercholesterolemia, which lack low density lipoprotein receptors. These results indicate that two lipoproteins that have been associated with athersclerosis--low density lipoprotein in humans and HDLc in cholesterol-fed swine--both can cause the accumulation of cholesterol and cholesteryl esters within cells through an interaction with the low density lipoprotein receptor.     

Effects of AY 9944 on low density lipoprotein metabolism in cultured human fibroblasts

Treatment of cultured human fibroblasts with the hypocholesterolemic drug AY 9944 resulted in a marked increase in low density lipoprotein internalization and degradation for concentrations up to 5 X 10(-6)M. Low density lipoprotein binding was less affected. Concentrations above 5 X 10(-6)M resulted in a relative decrease in low density lipoprotein degradation, whereas binding and internalization plateaued. The stimulation of low density lipoprotein internalization took place within the first hours of incubation of cells with the drug, which suggests a direct effect on the cell membrane. Such phenomenon could account at least partially for the hypocholesterolemic effect of the drug, besides its inhibitory effect on 7-dehydrocholesterol reductase.     

Functional expression of human and mouse low density lipoprotein receptors in hybridomas

Though a mouse.human-human heterohybridoma, N12-16.63, secreting an antitetanus toxoid human monoclonal antibody grew well in a serum-free medium, its high producing subclone N12-69 required SSGF-I, a low density lipoprotein (LDL) from swine serum, or human-LDL (h-LDL) for growth. The growth-promoting action of SSGF-I was caused by its lipid fraction, and SSGF-I could be replaced completely with cholesterol in the presence of bovine serum albumin (BSA). Thus, cell line N12-69 is a cholesterol auxotroph of the heterohybridoma. N12-69 cells express both mouse and human LDL receptors on the cell surface in a ratio of 1:4. SSGF-I bound to both receptors with the same binding affinity, and h-LDL was also take up by the same receptors, though the affinity constant of the receptors for SSGF-I was 1.5 times stronger than that for h-LDL. The growth of N12-69 cells was completely inhibited by the addition of dextran sulfate, which is known to inhibit the binding of LDL to LDL receptors, to an SSGF-I or h-LDL containing medium but was not inhibited at all when dextran sulfate was added to a serum-free medium supplemented with cholesterol and BSA. Furthermore, an anti-human LDL receptor monoclonal antibody partially inhibited the growth of N12-69 cells in an SSGF-I or h-LDL containing medium. These findings suggest that N12-69 cells express both biologically active mouse and human LDL receptors on their cell surfaces and that SSGF-I or h-LDL is taken up by the both receptors to be utilized as a cholesterol source for the growth.     

Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts

The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors.     

PAF-acether decreases low density lipoprotein degradation and alters lipid metabolism in cultured human fibroblasts

A 24 h pretreatment of human cultured fibroblasts with PAF-acether (PAF) induced a decrease in LDL degradation and a correlative accumulation of undegraded LDL. LDL binding was not significantly affected. Sterol and triacylglycerol synthesis from sodium acetate was enhanced whereas phospholipid synthesis decreased. Oleic acid incorporation into cholesteryl ester was markedly inhibited, whereas incorporation into triacylglycerols was increased. A decrease in the percentage of phosphatidylcholine and an increase in the percentage of phosphatidylethanolamine were found using sodium [32P]orthophosphate as precursor. These effects of PAF on LDL and lipid metabolism could be related to perturbations in membrane structure characteristics, leading to a delay in LDL delivery to lysosomes, and to modification of the activity of some key enzymes of lipid metabolism.     

Oxidized low density lipoprotein and innate immune receptors

PURPOSE OF REVIEW: Atherosclerosis is now recognized as a chronic inflammatory disease. This review discusses recent literature reporting that innate immune receptors bind oxidatively modified LDL and its many oxidized moieties and consequently modulate the atherogenic process. These innate pattern recognition receptors are known to play a central role in pro-inflammatory responses to bacteria by binding pathogen-associated molecular patterns. It is hypothesized that oxidized LDL exposes similar molecular patterns recognized by receptors of innate immunity. RECENT FINDINGS: Minimally modified LDL and its oxidized phospholipids have been found to bind to CD14 or activate Toll-like receptors on macrophages. In turn, various biological activities have been induced, including the stimulation of cytoskeletal rearrangements that alter phagocytic activity and the stimulation of cytokine secretion, such as IL-8. These findings link modified LDL with innate pattern recognition receptors, such as those involved in the lipopolysaccharide signaling pathway. Human epidemiological studies support the involvement of CD14 and TLR4 in cardiovascular diseases. Oxidized LDL has also been demonstrated to bind to C-reactive protein, an opsonic molecule activating classic complement pathway and Fcgamma receptor endocytosis. These data suggest that C-reactive protein may not only be a strong predictor of clinical disease, but may also play a role in atherogenesis. Recent data on other innate immune receptors are discussed in the context of their potential interactions with oxidized LDL and atherogenesis. SUMMARY: Recent findings suggest that oxidized forms of LDL interact with innate immune receptors. Further studies are needed to identify the role of these interactions in inflammation and atherosclerosis.     

Compartmentation and turnover of the low density lipoprotein receptor in skin fibroblasts

The low density lipoprotein receptor (LDLR) was immunoprecipitated from [35S]methionine-labeled skin fibroblasts derivatized at 4 or 18 degrees C with an impermeant biotinylating reagent. Separation of derivatized and underivatized receptor from immunoprecipitates by selective binding to streptavidin-agarose allowed assessment of receptor protein cellular compartmentation and rates of intercompartmental transfer. At both 4 and 18 degrees C the amount of LDLR that is derivatized in cells labeled to near steady state saturates after 1-2 h of reaction at, respectively, 47 and 70% of total immunoprecipitable receptor protein. On the basis of temperature titration experiments, protein exposed only to the cell surface reacts at 4 degrees C; raising the temperature of biotinylation to 18 degrees C provides access to an additional pool of receptor protein. Remaining LDLR is derivatized at 37 degrees C. LDLR unreactive at 18 degrees C largely resides in membrane compartment(s) devoid of plasma membrane on the basis of its fractionation on Percoll gradients. While total cellular LDLR and 4 degrees C-derivatized LDLR labeled to steady state turn over in a first order manner (t1/2 = 12-13 h), the specific activity of pulse-labeled, 4 degrees C-accessible protein peaks after 1-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.