Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

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

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

Sphingolipid-induced enhancement of receptor-mediated uptake of low density lipoproteins in normal and receptor-deficient human skin fibroblasts

(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides G_M1 or G_D1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced supression of [¹⁴C]acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of α₂-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.     

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

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

Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes.

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.     

Regulation of low-density lipoprotein metabolism by cell density and proliferative state

Stimulation of the proliferation of human skin fibroblasts by platelet-derived growth factor increased the binding and degradation of low-density lipoproteins at cell densities of 2000-30,000 cells/cm2. Binding and degradation of low-density lipoprotein was an inverse function of cell density in both proliferating and quiescent cells, indicating that the effect of cell density on the LDL receptor has proliferation-dependent and proliferation-independent components. The effect of medium conditioned by confluent fibroblasts on LDL metabolism was tested to determine if the effects of cell density on LDL metabolism might be mediated by cellular secretion products. Fibroblast-conditioned medium increased LDL metabolism, suggesting secretion products do not mediate these effects of cell density. These data indicate that regulation of the low-density lipoprotein receptor is not a simple on/off response to growth stimulation, but is responsive to extracellular cues such as cell density.     

Effect of sex hormones on human T cell activation by concanavalin A

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

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

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

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Age dependent production of a competence factor by human fibroblasts

Several cell types such as Balb/c 3T3 have been shown to require platelet-derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin-C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12-wk embryo, newborn, and 3-yr-old were exposed to serum-free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3-yr-old cells with the addition of plasma alone, whereas conditioned medium from the 3-yr-old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3-yr-old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3-yr-old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age-dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.     

Stimulation of proliferation of bovine placental cells by products of activated mononuclear leukocytes

Summary Culture medium conditioned with concanavalin A-stimulated mononuclear leukocytes was tested for its ability to stimulate in vitro proliferation of bovine placental cells. The crude preparation of cytokines caused a dose-dependent increase in [³H]thymidine uptake into cells obtained by trypsinization of fresh bovine placentae and placental cell lines established from cellular outgrowths of long-term bovine placental cultures, but had no effect on growth of 3T3 fibroblasts. Growth of trypsinized placental cells was not enhanced by culture in the presence of interleukin-2, interferon-β₂, interferon-γ, or tumor necrosis factor-α. These results corroborate those of murine studies, suggesting a growth-promoting role for cytokines released into the maternal-fetal interface. Supported in part by a grant from th e Florida Dairy Farmers Milk Checkoff Fund and a grant from CIBA-GEIGY. This is Journal Series R-01079 of the Florida Agricultural Experiment Station.     

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

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

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells.

Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M.     

Calmodulin antagonists stimulate LDL receptor synthesis in human skin fibroblasts

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.     

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

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