Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts

In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.     

Low density lipoprotein receptor binding in aging human diploid fibroblasts in culture

High affinity cell surface receptors for low density lipoproteins (LDL) are inducible in cultured human lung fibroblasts by the removal of lipoproteins from the cell culture medium. The binding, uptake, and degradation of 125I-LDL by fibroblasts decrease with increasing number of population doublings. The affinity of LDL receptor binding, however, remained unchanged at different population doublings levels. Hence, the difference in LDL binding activity in the aging fibroblasts can be attributed to a reduction in the number of receptor sites on the cell membrane. Cellular uptake of [4-14C]cholesterol and 2-deoxy-D-[1-14C]glucose mediated through mechanisms independent of the LDL receptor pathway revealed no significant difference in early and late passage fibroblasts. This suggests that the alteration in the LDL receptor binding in serially passaged fibroblasts is an "age-related" phenomenon. The late population doubling fibroblasts require more LDL in the culture medium for feedback inhibition of LDL receptor synthesis. Thus, aging fibroblasts are both progressively less inducible and less suppressible in the regulation of their cell membrane LDL receptors. Similar results were also obtained with respect to the regulation of DL-3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the aging fibroblasts in culture; the enzyme has become less inducible and less supressible as the fibroblasts approach the limit of their in vitro lifespan. These age-related alterations in the cellular metabolism of LDL and cholesterol might contribute to our understanding of the increased risk of athlerosclerosis in our aging population.     

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation by the oncogene v-fms: The effects of castanospermine on transformation-related parameters

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.     

Low density lipoprotein receptors on epithelial cell (Madin-Darby canine kidney) monolayers. Asymmetric distribution correlates with functional difference

Low density lipoprotein (LDL) receptors are present on both the apical and basal surfaces of confluent monolayers of Madin-Darby canine kidney (MDCK) epithelial cells grown on gelatin-coated polycarbonate filters. Although there is only a single species of receptor protein present, as shown by immunoblotting, the receptors on the two surfaces were found to behave differently. LDL receptors on the basal surface show all of the characteristics of the LDL receptor described in fibroblasts in that their number is dependent upon the sterol (or LDL) content of the medium; however, regulation is only affected by LDL in the medium in contact with the basal side. In contrast, the apical surface LDL receptors are not regulated by the presence of LDL in the media on either the apical or basal surface. LDL particles can be transported across the monolayer in a temperature-dependent and -specific manner from the apical to the basal sides of the cell, but not in the opposite direction. The binding of 125I-LDL to both surfaces can be effectively inhibited not only by unlabeled LDL and very low density lipoprotein, but also by an antibody directed against the LDL receptor. The data suggest that the LDL receptors on the two aspects of the cell surface are biochemically identical, but differ in function. Thus, the basal surface receptor is involved in the control of cell cholesterol homeostasis, while that on the apical surface is responsible for the transport of LDL to the basal side.     

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.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Soluble LDL-R are formed by cell surface cleavage in response to phorbol esters.

A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4beta-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins.     

Kifunensine inhibits glycoprotein processing and the function of the modified LDL receptor in endothelial cells

Kifunensine is an alkaloid that is produced by the actinomycete Kitasatosporia kifunense and resembles the cyclic oxamide derivative of 1-aminodeoxymannojirimycin in structure. We previously showed that this compound was a potent inhibitor of the purified glycoprotein processing enzyme, mannosidase I, and caused an almost complete inhibition in the formation of complex types of oligosaccharides with the concurrent accumulation of N-linked oligosaccharides having Man9(GlcNAc)2 structures in influenza virus-infected Madin Darby canine kidney cells. Kifunensine, at concentrations of 1 microgram/ml or higher in the culture medium, caused an almost complete inhibition in the formation of complex types of oligosaccharides by human skin fibroblasts or aortic endothelial cells, with the resulting accumulation of Man9(GlcNAc)2 oligosaccharides on the cell surface N-linked glycoproteins, and more specifically on the scavenger-LDL receptor. When endothelial cells were grown in the presence of 1 microgram/ml of kifunensine, there was a 75% inhibition in the ability of these cells to degrade 125I-labeled acetyl-LDL, but this inhibitor appeared to have little or no effect on the ability of either endothelial cells or fibroblasts to degrade 125I-labeled LDL, even at kifunensine concentrations of 10 micrograms/ml. Kifunensine also decreased the binding of the labeled acetyl-LDL by the scavenger receptor of the endothelial cells, but the amount of this inhibition relative to controls was significantly less than that of the degradation, suggesting that kifunensine affects two different steps of acetyl-LDL metabolism in these cells. Endothelial cells grown in the presence of 10 micrograms/ml of kifunensine had only half the activity of the lysosomal enzymes, beta-hexosaminidase, and proteases, as did control cells, although kifunensine did not affect [3H]leucine incorporation into protein. Thus, kifunensine apparently affects the activity of (some) lysosomal enzymes in an as yet undefined manner.     

Ammonium chloride causes reversible inhibition of low density lipoprotein receptor recycling and accelerates receptor degradation

The effects of the acidotropic agent, NH4Cl, on the recycling and turnover of low density lipoprotein (LDL) receptors were analyzed in human skin fibroblasts using ligand binding assays, [35S]methionine pulse-chase experiments, and electron microscopy. NH4Cl did not prevent receptor internalization but caused a marked redistribution of LDL receptors to intracellular sites (endosomes) that was completely dependent on the presence of apolipoprotein-B- or -E-containing ligands. Maximal inhibition of recycling was observed at LDL concentrations that only partially saturated receptors, suggesting that the receptors function as oligomers. In contrast, full receptor occupancy by the multivalent, apolipoprotein-E-containing beta-very low density lipoprotein was required for the same effect. The intracellular accumulation was reversible and the majority of receptors returned to the cell surface when NH4Cl was removed after short treatments. The rate of degradation of LDL receptors was greatly accelerated in the presence of NH4Cl and ligand, with a t1/2 of about 2 h (approximately 6 times faster than receptor degradation in the absence of NH4Cl). Neither the redistribution nor the accelerated loss of immunoprecipitable LDL receptors was observed in an LDL receptor internalization-defective mutant cell line. We conclude that NH4Cl inhibited the recycling specifically of occupied receptors, thereby accelerating their degradation, probably in endosomes.     

Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.     

Up-regulation of high density lipoprotein receptor activity by gamma-interferon associated with inhibition of cell proliferation

High density lipoprotein (HDL) binds to cell surface receptors and promotes selective removal of excess cholesterol from intracellular pools. The activity of this receptor is up-regulated when cells become loaded with cholesterol, but the relative degree of up-regulation depends on the growth state of the cells. The current study demonstrates that treatment of proliferating fibroblasts with gamma-interferon (IFN) increases the activity of the HDL receptor in association with a decrease in the rate of cell proliferation. Addition of IFN during the growth phase reduced the number of cells but had little effect on total cell protein, indicating that IFN inhibited cell proliferation but produced larger cells. IFN treatment increased the number of high affinity receptors for HDL on the surface of cholesterol-loaded fibroblasts, whether receptor binding was expressed per cell or per unit of cell surface area, cell protein, or cell cholesterol. IFN treatment also appeared to increase the amount of 110-kDa HDL binding protein in fibroblast membranes that has been postulated to represent the HDL receptor molecule. The IFN-induced increase in HDL receptor activity was associated with an enhanced ability of HDL3 to remove cholesterol from intracellular pools. These results are consistent with the hypothesis that inhibition of cell proliferation increases HDL receptor-mediated transport of excess cholesterol from cells, possibly to rid cells of cholesterol that accumulates in response to a reduced rate of membrane synthesis.     

Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts

Rat fibroblasts degraded human low density lipoprotein (LDL) very slowly, one-tenth to one-fortieth the rates observed in human fibroblasts. In rat cells, human LDL caused only very small increases in cell cholesterol content and acylCoA:cholesterol acyltransferase (ACAT) activity and caused only small decreases in beta-hydroxy-beta-methylglutaryl CoA (HMG CoA) reductase activity; in human cells, however, human LDL induced very large changes in all three of these parameters, as expected. The binding of human LDL to rat fibroblasts was not reduced by previous incubation with human LDL or with 25-hydroxycholesterol. Thus, in rat fibroblasts there appear to be few, if any, regulated high-affinity receptors that recognize human LDL. Rat LDL fractions (d 1.02-1.05 g/ml), in contrast, were degraded more rapidly than human LDL by rat fibroblasts, caused a significant increase in cell cholesterol content, an increase in ACAT activity, and a significant decrease in HMG CoA reductase activity. Moreover, the degradation of this rat LDL fraction by rat fibroblasts as a function of concentration was biphasic, i.e., there appeared to be a high-affinity component of degradation. Thus, it appears that rat fibroblasts do have a receptor for homologous lipoproteins. However, because both apoprotein B and apoprotein E are present in these rat lipoprotein fractions, the observed effects may relate to recognition of either or both of these apoproteins. The metabolism and metabolic effects of the conventionally defined high density lipoprotein (HDL) fraction of the rat by rat or human fibroblasts resembled those of human LDL in human fibroblasts. It is suggested that rat HDL may, because of its apo E content and higher concentration in rat plasma relative to that of LDL, play an important role in cholesterol homeostasis in vivo.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.     

Effect of preferential insertion of LDL receptors near coated pits

Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10 plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37°C.     

Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus.

In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

Effect of preferential insertion of LDL receptors near coated pits

Recent experiments suggest that low density lipoprotein (LDL) receptors on human fibroblasts are not inserted into the plasma membrane uniformly, as earlier experiments indicated, but are inserted into specialized regions, called plaques, where coated pits form. If the consequent reduction in the time required for LDL receptors to diffuse to coated pits were significant, this could alter conclusions drawn from previous calculations based on the assumption that LDL receptors are inserted uniformly. In particular, the conclusion could be wrong that diffusion of LDL receptors to coated pits is the rate limiting step in the interaction of cell surface LDL receptors with coated pits. Here we calculate the extent of the reduction in mean travel time of an LDL receptor to a coated pit, as a function of the plaque radius. We find that only if LDL receptor insertion is limited to a very small portion of the plasma membrane near coated pit sites is there a substantial decrease in the average time it would take an LDL receptor to diffuse to a coated pit. In order for preferential insertion of LDL receptors into plaques to cut the mean receptor travel time in half, plaques would have to take up no more than 10% of the cell surface area; to reduce the travel time by a factor of 10, plaques would have to cover only 2% of the cell surface, approximately twice the area covered by coated pits at 37 degrees C.     

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.