Intracellular Transport of Low Density Lipoprotein Derived Free Cholesterol Begins at Clathrin-Coated Pits and Terminates at Cell Surface Caveolae†

Biochemistry ◽  
1996 ◽  
Vol 35 (47) ◽  
pp. 14932-14938 ◽  
Author(s):  
Phoebe E. Fielding ◽  
Christopher J. Fielding
Keyword(s):  
Cell Surface ◽  
Free Cholesterol ◽  
Intracellular Transport ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Coated Pits

Platelet factor 4 binds to low-density lipoprotein receptors and disrupts the endocytic itinerary, resulting in retention of low-density lipoprotein on the cell surface

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3613-3622 ◽  
Author(s):  
Bruce S. Sachais ◽  
Alice Kuo ◽  
Taher Nassar ◽  
Jeanelle Morgan ◽  
Katalin Kariko ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Cultured Cells ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Platelet Factor 4 ◽  
Low Density ◽  
Coated Pits ◽  
Platelet Factor

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R–dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 μg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts

1988 ◽  
Vol 66 (2) ◽  
pp. 116-125
Author(s):  
Gayle A. Hoover ◽  
Suzanne McCormick ◽  
Norman Kalant
Keyword(s):  
Cell Surface ◽  
Human Skin ◽  
Free Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Collagen Gel ◽  
Skin Fibroblasts ◽  
Low Density ◽  
Human Skin Fibroblasts ◽  
Plastic Substratum

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na235SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13–25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 μg protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40–50% and of [35S]sulfate by 15–20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.

scholarly journals Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1156-1168 ◽  
Author(s):  
D J McGookey ◽  
R G Anderson
Keyword(s):  
Cholesteryl Ester ◽  
Lipase Activity ◽  
Lipid Droplets ◽  
Free Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
The Body ◽  
Cyclic Process ◽  
Low Density ◽  
Cholesteryl Esters

Mouse peritoneal macrophages can be induced to accumulate cholesteryl esters by incubating them in the presence of acetylated low density lipoprotein. The cholesteryl esters are sequestered in neutral lipid droplets that remain in the cell even when the acetylated low density lipoprotein is removed from the culture media. Previous biochemical studies have determined that the cholesterol component of cholesteryl ester droplets constantly turns over with a half time of 24 h by a cyclic process of de-esterification and re-esterification. We have used morphologic techniques to determine the spatial relationship of cholesteryl ester, free cholesterol, and lipase activity during normal turnover and when turnover is disrupted. Lipid droplets were surrounded by numerous 7.5-10.0-nm filaments; moreover, at focal sites on the margin of each droplet there were whorles of concentrically arranged membrane that penetrated the matrix. Histochemically detectable lipase activity was associated with these stacks of membrane. Using filipin as a light and electron microscopic probe for free cholesterol, we determined that a pool of free cholesterol was associated with each lipid droplet. Following incubation in the presence of the exogenous cholesterol acceptor, high density lipoprotein, the cholesteryl ester droplets disappeared and were replaced with lipid droplets of a different lipid composition. Inhibition of cholesterol esterification caused cholesteryl ester droplets to disappear and free cholesterol to accumulate in numerous myelin-like structures in the body of the cell.

scholarly journals Macrophages Create an Acidic Extracellular Hydrolytic Compartment to Digest Aggregated Lipoproteins

2009 ◽  
Vol 20 (23) ◽  
pp. 4932-4940 ◽  
Author(s):  
Abigail S. Haka ◽  
Inna Grosheva ◽  
Ethan Chiang ◽  
Adina R. Buxbaum ◽  
Barbara A. Baird ◽  
...  
Keyword(s):  
Free Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Critical Event ◽  
Low Density ◽  
Acid Hydrolases ◽  
Detailed Understanding ◽  
Physical Features

A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake. We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing of portions of the compartments, allowing formation of an “extracellular” proton gradient. An increase in free cholesterol is observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein. A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.

scholarly journals Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans.

1996 ◽  
Vol 184 (5) ◽  
pp. 1699-1711 ◽  
Author(s):  
M Shakibaei ◽  
U Frevert
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Heparan Sulfate Proteoglycans ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Related Protein ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells results in more than 90% inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin, or anti-LRP antibodies reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell invasion.

scholarly journals Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts

1985 ◽  
Vol 228 (1) ◽  
pp. 219-225 ◽  
Author(s):  
B B Lundberg ◽  
L A Suominen
Keyword(s):  
Free Cholesterol ◽  
Water Solubility ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Lung Fibroblasts ◽  
Cell Protein ◽  
Low Density ◽  
Free System

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.

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