Presence Of Low-density Lipoprotein Research Articles

Induction of platelet-derived growth factor chain a gene expression in human smooth muscle cells by oxidized low density lipoproteins

Abnormal proliferation of vascular smooth muscle cells is a key event in the formation of atherosclerotic plaques in humans. It has been suggested that modulation of platelet derived growth factor chain A (PDGF-A) gene expression may contribute to atherosclerosis. Using an 'in situ hybridization' technique the present study shows that the presence of low density lipoproteins induces an eight fold increased expression of the transforming gene PDGF-A in human smooth muscle cells. The influence on PDGF-A expression only occurred after oxidative modification of low density lipoproteins, a process known to be important in atherogenesis. The non-atherogenic high density lipoproteins did not modulate the PDGF-A mRNA levels in smooth muscle cells. Consequently, oxidized low density lipoproteins may participate in atherosclerotic lesion formation as a result of autocrine stimulation of PDGF-A.

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.

Activation of 15-lipoxygenase by low density lipoprotein in vascular endothelial cells. Relationship to the oxidative modification of low density lipoprotein

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12+15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

Photosensitization by Photofrin II delivered to WI26VA4 SV40-transformed human fibroblasts by low density lipoproteins: inhibition of lipid synthesis and fatty acid uptake.

Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.

Follicular fluid lipoproteins in preovulatory period and their relationship with follicular maturation and progesterone production by human granulosa-luteal cells in vivo and in vitro

Follicular fluid (FF) lipoprotein content was evaluated in an in vitro fertilization/embryo transfer and gamete--intrafallopian--transfer program and correlated to follicular and oocyte maturation. Moreover, the in vitro progesterone response of granulosa-luteal cells from 10 patients to high-density lipoprotein (HDL) and to low-density lipoprotein (LDL) was assessed. Most FFs contained only HDL. Sixteen out of 97 FFs contained also very low levels of LDL and very-low-density lipoprotein (VLDL). The presence of LDL was associated to features of follicle and oocyte hypermaturity. LDL alone induced a much more potent increase of progesterone (P) release by granulosa-luteal cells than HDL alone, and the HDL partially reversed the potent effect of LDL. It is concluded that in late follicular phase HDL maintains P release by granulosa cells at a low rate and prevents a potent stimulation of P production by LDL which might cross the maturating blood-follicle barrier, until increasing passage of LDL in FF decreases the HDL:LDL ratio and the action of LDL becomes prominent.

Compartmentalization of cholesterol metabolism and cellular growth in cultured intestinal crypt cells

Growth of rat intestinal crypt derived cells IEC-6 ceased when the key enzyme of cholesterol synthesis, hydroxymethylglutaryl-CoA reductase, was blocked by the competitive inhibitor mevinolin. This effect was reversed by the addition of mevalonolactone. LDL suppressed reductase activity as well as cholesterol synthesis from [14C]octanoate and stimulated acyl-CoA cholesterol acyltransferase, but failed to support cell growth despite rapid receptor mediated degradation even in the presence of low mevalonolactone concentrations. Inhibition of cholesterol esterification by Sandoz-Compound 58-035 enhanced cell growth in the presence of mevinolin, but did not promote proliferation in the additional presence of low-density lipoproteins. HDL3 but not HDL2 or tetranitromethane-modified HDL3 totally reversed the mevinolin induced inhibition of cell growth. This rescue by HDL3 was overcome by an increased dose of mevinolin. HDL3 derepressed reductase, stimulated cholesterol synthesis and reduced cholesterol esterification, but did not reverse the cholesterol synthesis inhibition by mevinolin. It is concluded that IEC-6 cells preferentially use endogenously synthesized cholesterol for membrane formation rather than low-density lipoprotein cholesterol. High-density lipoproteins appear to normalize cell growth in the presence of mevinolin by inhibition of cholesterol esterification and probably by inducing the formation of non sterol products of mevalonate.

Regulation of the uptake of high-density lipoprotein-originated cholesteryl ester by HepG2 cells: Role of low-density lipoprotein and plasma lipid transfer protein

Cholesteryl ester uptake by the human hepatoma cell line HepG2 was studied in vitro by using radiolabeled cholesteryl ester as a tracer. After the cells were incubated in a lipoprotein deficient condition, the rate of radio labeled cholesteryl ester uptake from low-density lipoprotein (LDL) was estimated to be some 25-times higher than that from high-density lipoprotein (HDL). LDL-cholesteryl ester uptake was suppressed by preincubation of the cells with LDL, but pretreatment of the cells with HDL did not show significant effect. HDL-cholesteryl ester uptake was only slightly suppressed by pretreatment of the cells with LDL, and there was no effect with HDL pretreatment. HDL-cholesteryl ester uptake was not affected either by the presence of LDL or human plasma lipid transfer protein alone in the medium under our experimental conditions. Lipid transfer protein enhanced the uptake of radiolabeled cholesteryl ester originating from HDL by the cells only in the presence of LDL. Thus, lipid transfer protein catalyzes a bypass to LDL for the uptake by HepG2 cells of cholesteryl ester molecules which originate in HDL, and this pathway is much more efficient than direct uptake of cholesteryl ester originating in HDL by these cells.

Effect of prostaglandin E 1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro

The effect of PGE 1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in huna, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1–6 μg protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE 1-concentrations (100 pM to 100 μM). In separate experiments the effect of PGE 1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p<0.001) lower binding capacity for 123I-LDL. PGE 1 significantly (p<0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and th binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE 1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to specific apo-B-receptors in a dose-dependent manner (p<0.01–0.001) with an ED 50 of 123±64 nM for human liver, 90±102 nM for rat liver obtained during anasthesia, 74±23 nM for rat liver obtained after decapotation and 941±121 nM for swine liver. In human liver iloprost (ED 50=876±53 nM) and PGI 2 (ED 50=52±12 nM) were less effective than PGE 1, PGE 2 had no effect on LDL-induced competition. It is concluded that PGE 1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE 1.

Effect of a novel squalene epoxidase inhibitor, NB-598, on the regulation of cholesterol metabolism in Hep G2 cells

We have reported previously that NB-598 competitively inhibits human squalene epoxidase and strongly inhibits cholesterol synthesis from [14C]acetate in cultured cells. Furthermore, multiple oral administration of NB-598 decreased serum cholesterol levels in dogs (Horie, M., Tsuchiya, Y., Hayashi, M., Iida, Y., Iwasawa, Y., Nagata, Y., Sawasaki, Y., Fukuzumi, H., Kitani, K., and Kamei, T. (1990) J. Biol. Chem. 265, 18075-18078). In the present study, the effects of NB-598 on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density-lipoprotein (LDL) receptor were examined using a human hepatoma cell line Hep G2. Incubation of Hep G2 cells with NB-598 for 18 h increased HMG-CoA reductase activity in a dose-dependent manner. However, the increase in activity induced by NB-598 was lower than that induced by L-654,969 (a potent HMG-CoA reductase inhibitor), although NB-598 inhibited cholesterol synthesis more potently than L-654,969. On the other hand, HMG-CoA reductase mRNA was increased to the same extent by both inhibitors. These results demonstrate that NB-598 does not inhibit the synthesis of non-sterol derivative(s) of mevalonate, which regulate HMG-CoA reductase activity at the post-transcriptional level. NB-598 increased the binding of 125I-LDL to Hep G2 cells. LDL receptor mRNA was also induced by NB-598. In the presence of LDL or cycloheximide, NB-598 did not increase LDL receptor activity. These results demonstrate that the induction of LDL receptor activity by NB-598 is due to increases in mRNA and protein through the inhibition of cholesterol synthesis at the squalene epoxidase step. From these observations, squalene epoxidase inhibitor is expected to be highly effective in the treatment of hypercholesterolemia and also is very useful as a research tool for studying the regulation of cholesterol metabolism.

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.

The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum.

A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, beta-galactosidase of Escherichia coli, has been previously constructed. This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its beta-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this report, we demonstrate that the loss of beta-galactosidase activity results from the accelerated degradation of the HMGal protein. Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection. Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold. Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism. This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein. In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity. Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum.

Uptake of the aromatic retinoids Ro 10-9359 (etretinate) and Ro 10-1670 (acitretin), its main metabolite, delivered to cultured human fibroblasts by serum proteins. Lack of evidence for perturbation of LDL catabolism

Etretinate or acitretin are efficiently delivered to cultured human fibroblasts in the presence of low density lipoproteins, high density lipoproteins or human serum albumin. In contrast to acitretin, delivery of etretinate to fibrobasts is more efficiently achieved with human serum albumin than with lipoproteins. The uptake of etretinate and acitretin via low density lipoproteins delivery, does not take place via the low density lipoprotein-receptor endocytotic pathway but mostly through a passive exchange with the plasma membrane. However, in contrast to acitretin, the exchange of etretinate seems to occur alter binding of etretinate-loaded low density lipoproteins to the apolipoprotein B receptors. No differences are observed in binding, internalization and degradation of native, etretinate-loaded low density lipoproteins and acitretin-loaded low density lipoproteins, suggesting that the presence of these retinoids in low density lipoproteins does not alter their processing by the cells. Furthermore, the presence of these retinoids in the cells does not notably affect, under our experimental conditions, the catabolism of native low density lipoproteins.

Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.

Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.

Enhancement of Thyroxine Entry into Low Density Lipoprotein (LDL) Receptor-Competent Fibroblasts by LDL: An Additional Mode of Entry of Thyroxine into Cells

Having demonstrated that plasma low density lipoproteins (LDL) bind T4 through a specific interaction with their sole apolipoprotein, apoB-100, we tested the hypothesis that cells could internalize the LDL-T4 complex via cell surface LDL receptors. These receptors are down-regulated by cholesterol loading and up-regulated by cholesterol deficiency. We, therefore, studied the uptake of [125I]T4 or [125I]T3 by human skin fibroblasts grown in 10% lipoprotein-deficient serum in the absence or presence of LDL. At concentrations of LDL (12.5 and 22 micrograms protein/ml) that gave significant binding of T4 but did not exceed LDL receptor capacity, both the initial rate of saturable T4 uptake and the uptake at equilibrium increased by 27-63%. No significant increase occurred at a LDL concentration of 1.6 micrograms protein/ml (less than 3% occupancy), whereas there was a 20 to 31% reduction at 125 micrograms/ml (approximately 5 times the saturation dose for the LDL receptor). These changes were confirmed with several different LDL preparations and were mimicked by isolated apoB-100 and apoE, the sole ligands for the LDL receptors (apoB/E receptors). T4 uptake did not increase in normal fibroblasts with down-regulated LDL receptors or in LDL receptor-deficient fibroblasts from a patient with familial hypercholesterolemia. In the latter cell line the uptake of T4 (and T3) in the absence of LDL was indistinguishable from that of normal fibroblasts. T3 uptake in normal fibroblasts was not enhanced by LDL. The specificity of the LDL effect was shown by the finding that T4-binding globulin, prealbumin, or serum albumin, at concentrations giving 10-90% T4 bound, failed to increase T4 uptake. Instead, each of these major thyroid hormone-binding plasma proteins caused a dose-dependent decrease in T4 entry. It is concluded that at least two modes of entry into fibroblasts are available for T4. The first is the cell surface thyroid hormone-binding sites, which recognize the free hormone and are present in both normal and LDL receptor-negative fibroblasts. The second, and additional, mode of entry is via the LDL receptors, which recognize the T4-LDL complex, are absent in LDL receptor-negative fibroblasts, are reduced in down-regulated fibroblasts, and are unavailable for T3, owing to the low affinity of T3 for LDL.

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase.

In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.

Differential stimulation of growth related metabolism in cultured smooth muscle cells from SHR and WKY rats by combinations of EGF and LDL

We have reported previously that vascular smooth muscle cells from spontaneously hypertensive rats (SHR) were more responsive to epidermal growth factor (EGF) than their normotensive derived Wistar Kyoto (WKY) controls. This differential responsiveness is evident for several cellular processes including activation of S 6-kinase, elevation of intracellular pH and stimulation of both phosphoinositide metabolism and DNA synthesis. Quiescent smooth muscle cells exposed to low density lipoprotein (LDL) exhibited a similar differential responsiveness (SHR > WKY) in terms of S 6-kinase activation, which was time- and dose-dependent (10 −10–10 7 M), but neither cell type responded appreciably to LDL in terms of a stimulation in [ 3H]-thymidine incorporation. Exposure of the same cells to EGF and LDL in combination elicited a marked synergistic stimulation in DNA synthesis, the extent of which was greater for SHR than WKY. The sensitivity of both cell types to EGF was increased in the presence of LDL, although cells from hypertensive animals still exhibited their greater (vs. WKY) sensitivity. In both cell types, activation of nuclear protooncogenes c-fos and c-myc by LDL was minimal, whereas oncogene induction by EGF was ∼ five-fold greater for SHR-derived cells compared to those from WKY animals. No marked synergistic effect on the time-dependent induction of either entity was observed for cells exposed to EGF and LDL simultaneously, and the response of SHR-cells remained greater than WKY-cells.

Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up- or down-regulation

Treatment of cultured human skin fibroblasts with cycloheximide retarded the down-regulation of low density lipoprotein (LDL) receptor activity caused by 25-hydroxycholesterol. The rate of LDL receptor degradation, measured directly by means of [35S]methionine pulse-chase experiments, was also markedly inhibited by cycloheximide (or puromycin), suggesting that continuous synthesis of a short-lived mediator protein(s) was necessary for normal LDL receptor turnover. In the absence of cycloheximide, both the up- and down-regulation of LDL receptor activity took place with a half-time of approximately 12 hr. Pulse-chase measurements with [35S]methionine yielded a receptor half-life (t1/2) of 11.7 +/- 2.2 hr (n = 10) in up-regulated cells; the t1/2 in the partially down-regulated state was similar. The presence of LDL or 25-hydroxycholesterol did not alter this degradation rate. Regulation of LDL receptor activity under these various culture conditions therefore probably occurred solely as a result of changes in the rate of receptor synthesis. The cycloheximide-sensitive factor(s) that influences receptor turnover apparently did not play a regulatory role in the up- or down-regulation of the LDL receptor.

Defective utilization of cholesterol esters from low-density lipoprotein in a human acute lymphoblastic leukemia T cell line

The glucocorticoid sensitive CEM-C7 T cell line was derived from human acute lymphoblastic leukemia cells by Norman and Thompson (Cancer Res. 37 (1977) 3875). Madden et al. (Cancer Res 46 (1986) 617) have demonstrated that the growth inhibitory effect of glucocorticoids on these cells is due in part to an inhibition of cholesterol synthesis even in the presence of low-density lipoprotein (LDL)-containing serum. To delineate further the role of cholesterol in this growth inhibition, we have examined the ability of LDL-bound [ 3H]cholesterol linoleate to reverse the growth inhibitory effect of 1 μM dexamethasone on the CEM-C7 cells. LDL-bound cholesterol linoleate did not reverse the dexamethasone-mediated growth inhibition. Although incorporation of [ 14C]acetate into free cholesterol was inhibited only 20% by incubation with LDL, the presence of dexamethasone further inhibited acetate incorporation into free cholesterol in the LDL-treated cells. Dexamethasone had no effect on the uptake or utilization of LDL-bound cholesterol linoleate. Under all conditions, more than 99% of the acetate incorporated into cholesterol was present as free cholesterol, while over 80% of the LDL-derived cholesterol linoleate remained in the ester compartment. In contrast, in normal lymphocytes, over half the LDL-derived cholesterol was converted to free cholesterol. Direct analysis of the acid cholesterol ester hydrolase, the enzyme primarily responsible for processing LDL-bound cholesterol esters, revealed over 60-times the activity of this enzyme in the normal lymphocytes as compared to the activity present in the C7 cells. This finding cautions against the assumption that the presence of lipoprotein-containing serum provides an adequate, usable source of cholesterol for all cells.

Effects of in vivo and in vitro administration of prostaglandin F2 alpha on lipoprotein utilization in cultured bovine luteal cells.

Two experiments were conducted to determine if a loss in the ability to utilize lipoprotein-cholesterol is one mechanism whereby prostaglandin F2 alpha (PGF2 alpha) decreases steroidogenesis in bovine luteal cells. In the first experiment, serum-free cultures of bovine luteal cells were treated with PGF2 alpha (100 ng/ml) for 5 days prior to addition of lipoproteins. Exposure to PGF2 alpha completely suppressed low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-stimulated progesterone production (p less than 0.01) compared to control (no PGF2 alpha) cultures. Luteal cells cultured in the presence of LDL + luteinizing hormone (LH, 10 ng/ml) + PGF2 alpha produced significantly less progesterone than luteal cells cultured with LDL + LH (p less than 0.05). Treatment with PGF2 alpha had no significant effect on HDL + LH-stimulated progesterone synthesis. In the second experiment, cows were injected with a luteolytic dose of PGF2 alpha (25 mg), and the corpora lutea were removed at 0 (no PG), 1, 4, or 12 h post-injection. Dissociated luteal cells were placed in culture for 7 days, either with or without LH (10 ng/ml), and lipoproteins were added on Days 5-7. LH stimulation of progesterone production was apparent in cultures obtained at 0 and 12 (p less than 0.05) but not 1 and 4 h post-PGF2 alpha. Addition of either LDL or HDL increased progesterone synthesis in all cultures, regardless of time following in vivo administration of PGF2 alpha. It is concluded that PGF2 alpha can inhibit bovine luteal cell utilization of either LDL or HDL in vitro. However, luteal cell utilization of lipoproteins in vitro is not adversely affected by in vivo exposure to PGF2 alpha, if collected within 12 h post-PGF2 alpha.