Effect of cyclic AMP on low density lipoprotein binding and internalization by cultured human fibroblasts

Pretreatment of cultured human fibroblasts by cyclic AMP resulted in a marked decrease in the binding and internalization of the low density lipoproteins (about 55% of controls for cyclic AMP 2.10(-3) M). This effect was dose dependent and increased by theophyllin. DL propranolol, an inhibitor of adenylcyclase, had an opposite effect. Isoproterenol, which stimulates adenylcyclase, reproduced the effect of cyclic AMP. The cholesterol synthesis from [2-14C] acetate was decreased by cyclic AMP, theophyllin and isoproterenol, and increased by propranolol. The incorporation of [1-14C] oleate into cholesteryl esters was reduced by cyclic AMP, theophyllin, isoproterenol and propranolol.     

Involvement of the GTPase Rho in the cellular uptake of low density lipoprotein by human skin fibroblasts

Members of the Rho subfamily of small GTPases have been implicated in the regulation of endocytosis of ligand/receptor complexes localised to clathrin-coated pits. In this paper, we investigated the role of Rho A in the post-receptor regulation of cellular uptake and metabolism of native low density lipoprotein (LDL) by primary human skin fibroblasts. Incubations of cells with the selective Rho GTPase inhibitor C3-transferase (C3T) upregulated the binding, lysosomal degradation and cell accumulation of labelled LDL. The rate of internalisation of surface-bound LDL was also higher in C3T-treated cells. Single cell injections with C3T and dominant active V14Rho confirmed the negative regulation of LDL uptake by Rho. While cells injected with C3T internalised more 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL, diI-LDL internalisation was dramatically suppressed in cells injected with the constitutively active V14Rho. The negative regulation of LDL uptake by Rho appeared to be independent of changes in the actin cytoskeleton. An increasing number of naturally occurring toxins and serum factors have been shown to influence Rho GTPase signalling cascades. The herein described post-translational regulation of LDL internalisation may modulate cell events occurring subsequent to cellular lipoprotein uptake.     

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.     

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.     

Differential uptake of proteoglycan-selected subfractions of low density lipoprotein by human macrophages

Macrophages and arterial chondroitin sulfate proteoglycans (CSPG) are probably associated with extracellular and intracellular lipoprotein deposition during atherogenesis. We found that human arterial CSPG can be used to select subclasses from low density lipoprotein (LDL) with different structural properties and capacities to interact with human monocyte-derived macrophages (HMDM). Four subclasses, LDL(PG)1 to LDL(PG)4, in order of decreasing CSPG-complexing capacity, were prepared and characterized in terms of their ability to interact with HMDM. The LDL subclasses with highest avidity for CSPG, LDL(PG)1 and LDL(PG)2, were bound, internalized, and degraded more efficiently than those of lower avidity for CSPG. From LDL(PG)1 to LDL(PG)4, the gradual decrease in uptake by HMDM and decreasing avidity for CSPG were associated with a gradual decrease in isoelectric point (from 5.93 to 5.68) and an augmented ratio of surface polar lipid to core nonpolar components (from 0.35 to 0.54). Competition experiments indicated that the proteoglycan-selected subfractions shared the binding sites and uptake mechanisms of native LDL. The results suggest the existence of a structurally related gradation in the avidity of LDL subpopulations for cells and matrix components. The presence within LDL subpopulations of a differential capacity to interact with intimal extracellular and cellular elements could be associated with a similar heterogeneity in their atherogenic potential.     

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.     

Effects of dichloroacetate and glyoxylate on low density lipoprotein uptake and on growth of cultured fibroblasts

The effects of dichloroacetate, a known hypocholesterolemic agent, were studied in cultured growing and confluent human fibroblast cells. Microscopic examination showed no visible adverse effects of dichloroacetate on confluent cells during exposure to concentrations as high as 5 mM for 96 hr. Higher concentrations resulted in cell death after varying periods of incubation. There were no viable cells after 24 hr of exposure to 100 mM dichloroacetate. In contrast, much lower concentrations proved lethal to growing cells; cell growth, as determined by cell numbers at specified times after splitting, was suppressed by 1 mM dichloroacetate and 5 mM concentrations resulted in cell death. Similar effects were noted with glyoxylate. The hypocholesterolemic effect of dichloroacetate is probably not due to any effect on the low density lipoprotein pathway, since concentrations of up to 1 mM dichloroacetate did not affect the cellular binding and uptake of 125I-labeled low density lipoprotein. It is concluded that growing and rapidly metabolizing cells are much more sensitive to the toxic effects of dichloroacetate and glyoxylate than confluent cells.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

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.     

Enhanced binding of phospholipase-A2-modified low density lipoprotein by human adipocytes

Recognition of low density lipoprotein (LDL) by human adipocytes is not dependent on the classical LDL (apoprotein B-E) receptor. To assess whether LDL phospholipids have a role in adipocyte-LDL interactions, binding studies were carried out with human LDL modified with cobra venom phospholipase A2 (PLA2) and freshly isolated adipocytes and purified adipocyte plasma membranes prepared from surgical biopsies. LDL incubated with PLA2 showed increased monoacylphospholipid content, decreased diacylphospholipid content, and increased anodic migration on agarose gel electrophoresis. LDL cholesterol, triglyceride, and protein content remained unchanged. Typically, modification of 16 and 47% of LDL phospholipids enhanced specific binding of 125I-labelled LDL to plasma membranes progressively from 3.1 micrograms LDL bound/mg membrane protein (control) to 5.8 and 28.2 micrograms LDL bound/mg membrane protein, respectively. Nonspecific binding was not altered significantly. Excess unlabelled native LDL and high density lipoprotein (HDL3) effectively inhibited binding of PLA2-modified LDL. Freshly isolated adipocytes also showed enhanced binding and uptake of PLA2-modified LDL (0.1 vs. 0.9 micrograms LDL/10(6) cells x 2 h), control vs. modified). The results demonstrate that alterations of LDL phospholipids significantly enhance LDL binding and suggest a regulatory role for phospholipids in lipoprotein-cell interaction. Furthermore, the results support the view that human adipose tissue may be involved in the metabolism of modified lipoproteins, in vivo.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts.

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion. Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of 125I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on 125I-labelled LDL metabolism. Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of 125I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

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.     

Macrophage binding and the uptake of oxidized low density lipoprotein are regulated by intracellular protein phosphorylation

The involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosin light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Effect of manipulation of phospholipid polar head group on low density lipoprotein metabolism in human cultured fibroblasts

Modification of phospholipid polar head group was achieved by growing human cultured fibroblasts in medium devoid of serum and supplemented with N-methyl ethanolamine or N,N-dimethylethanolamine during 48 h. The corresponding phospholipids accounted for approximately 45% of total phospholipids. Whereas low density lipoprotein (LDL) binding was unaffected, LDL internalization and degradation appeared to be markedly reduced in the presence of N-methylethanolamine. N,N-dimethylethanolamine had no effect on the three studied parameters. These results emphasize the importance of phospholipid polar head group in LDL processing by receptor-mediated endocytosis.     

Human breast milk stimulates low density lipoprotein (LDL) binding to human skin fibroblasts in culture

Human breast milk incorporated at 1% concentration into the culture medium significantly (p less than 0.05) increased the binding of 125I-LDL to receptors of human skin fibroblasts in culture. Homogenized cows milk and infant formula (Similac) also possessed this stimulating property. The stimulating activity of milk persisted after dialysis and extraction with cold acetone. These preliminary studies suggest that milk might contain potent factor(s) influencing cholesterol metabolic process in early life.     

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.