A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

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.     

Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.     

First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins

The ligand binding domain of the low density lipoprotein receptor consists of seven cysteine-rich repeats of approximately 40 amino acids each. These repeats, which are located at the NH2 terminus of the protein, are homologous to sequences in complement components C8 and C9. To determine the role of the first repeat (amino acids 2-42), we prepared two plasmids containing expressible low density lipoprotein receptor cDNAs. The first plasmid, p delta R1, lacks only the nucleotides encoding the first repeat. It produced a receptor that bound and internalized lipoproteins and recycled to the cell surface with the same efficiency as the normal receptor. This deleted receptor failed to bind two monoclonal antibodies, IgG-C7 and IgG-15C8, which were shown previously to react with the ligand-binding domain. The second plasmid, pR1, encodes a markedly truncated protein whose extracellular domain consists of the first repeat joined to the transmembrane and cytoplasmic domains. This protein bound the two monoclonal antibodies with the same affinity as the normal receptor, but failed to bind lipoproteins. Binding of IgG-15C8 to the normal receptor and the pR1-encoded protein was Ca2+-dependent, indicating that the first repeat binds Ca2+. We conclude that repeats 2-6 in the ligand-binding domain are sufficient for binding lipoproteins and that the first repeat is highly immunogenic, but is not required for lipoprotein binding.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

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.     

Tissue factor pathway inhibitor inhibits endothelial cell proliferation via association with the very low density lipoprotein receptor

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new anti-angiogenic and antitumor agents.     

Gradient acrylamide/agarose gels for electrophoretic separation of intact human very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins

An exponential gradient gel with 0-10% acrylamide and 0.5% agarose was developed for electrophoresis of intact high molecular weight lipoproteins. This system resolves very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins in a size-dependent fashion. The characteristic relative mobility of these species can be determined in relation to protein and colloidal gold reference materials. Electron microscopy of selected lipoprotein fractions confirmed that relative mobility was related to apparent lipoprotein diameter. The composite gel medium can be used with prestained lipoproteins and permits immunoelectroblotting for qualitative analysis of apolipoprotein constituents.     

Lysophosphatidic acid stimulates phospholipase D activity and cell proliferation in PC-3 human prostate cancer cells

Phospholipase D (PLD) is activated in mammalian cells in response to a variety of growth factors and may play a role in cell proliferation. Lysophosphatidic acid (LPA) is a bioactive metabolite potentially generated as a result of PLD activation. Two human prostate cancer cell lines, PC-3 and LNCaP, express membrane PLD activity. The effects of LPA on PLD activity and proliferation were examined in PC-3 cells, which express hPLD1a/1b. Phorbol 12-myristate 13-acetate (PMA) induced a prolonged activation of PLD, as detected in both intact cells and membranes. LPA induced a transient activation of PLD that was maximal by 10 minutes. The EC₅₀ for LPA-induced PLD activation was approximately 1 μM. Pertussis toxin did not inhibit activation of PLD by LPA or PMA. Ro-31-8220 and bisindolylmaleimide I, inhibitors of protein kinase C, blocked activation by PLD by both PMA and LPA. PMA-induced activation of PLD did not appear to require translocation of PLDs from cytosol to membrane. LPA stimulated proliferation of PC-3 cells with an EC₅₀ of approximately 0.2 μM; this response was not inhibited by pertussis toxin. Perillyl alcohol, an anti-cancer drug, reversibly inhibited proliferation in response to either serum or LPA but did not inhibit activation of PLD by PMA or LPA. These data establish that LPA activates PLD and stimulates proliferation via G_i-independent pathways in a human prostate cancer cell line. J. Cell. Physiol. 174:261–272, 1998. © 1998 Wiley-Liss, Inc.     

Binding of low density lipoproteins to lipoprotein lipase is dependent on lipids but not on apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.     

Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.

Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed.     

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

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

The determination of the potential anticancer effects of Coriandrum sativum in PC-3 and LNCaP prostate cancer cell lines

Coriander (Coriandrum sativum L.) is such an herb from the Apiaceae family, used both for its medicinal and nutritional properties for many centuries. In this study, the effects of C. sativum extract on gene expression, viability, colony formation, migration, and invasion of PC-3 and LNCaP prostate cancer cell lines have been investigated. The half maximal inhibitory concentration (IC₅₀) dose in PC-3 and LNCaP cells was detected to be 2 and 5 mg/mL at the 24th hour, respectively. C. sativum extracts have been observed to cause a significant decrease in the expression of Akt and Bcl-2 in the PC-3 cells and just Akt in LNCaP cells while increasing in the expression of p53, caspase-9, caspase-10, PTEN, DR5, TRADD, PUMA, and NOXA. DR4 expression was increased in LNCaP cell line but not PC-3, and APAF and BID had increased expression in PC-3 but not the LNCaP cells. Our observations have shown that C. sativum extract decreased colony formation while inhibiting cell invasion and migration. Cell migration was hindered in PC-3 but not the LNCaP cells. In conclusion, this data present a valuable addition to the very limited data available out there on the potential use of C. sativum in prostate cancer treatment.