Cholesterol ester concentrations and the ester hydrolases in cultured glioblastoma cells: Effect of lipoprotein depleted serum

We have investigated the effects of substituting lipoprotein depleted serum (LPDS) for normal fetal calf serum (FCS) in culture media on cholesterol ester concentrations and the activity of the ester hydrolases in cultured glioblastoma (C-6 glial) cells. Glial cells grown in media supplemented with 10% FCS contained 16–23% of total cholesterol as esterified sterol. Total sterol content of the cells cultured in media supplemented with LPDS was reduced by 55–75% as compared to cells cultured in FCS media and none of this sterol was in esterified form. Cholesterol ester hydrolase activity was maximal at pH values of 4.5 and 6.5 and required Triton X-100 for optimal activity. Cholesterol ester hydrolase activity at pH 4.5 was significantly higher in cells grown in FCS media than in cells cultured in LPDS media, but the activity at pH 6.5 was not significantly different. The protein: DNA ratio of cells cultured in FCS was higher than in cells cultured in LPDS. These findings indicate that the increase in cholesterol ester concentrations in cells is accompanied by increased activity of lysosomal cholesterol ester hydrolase; and suggest that, in cells cultured in FCS, the availability of free cholesterol for incorporation into cellular membranes is regulated by cholesterol ester hydrolase. The findings also indicate that changes in growth and differentiation of cells cultured in LPDS may be related to reduced availability of exogenous cholesterol.     

Marked induction of sterol 27-hydroxylase activity and mRNA levels during differentiation of human cultured monocytes into macrophages

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.     

Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells

Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol synthesis by cholesterol sulfate in cultured fibroblasts

Although widely distributed throughout mammalian tissues, the biological function of cholesterol sulfate remains largely unknown. In these studies we have demonstrated that cholesterol sulfate suppresses de novo sterol synthesis in cultured human fibroblasts. It was further shown in these cultured cells that cholesterol sulfate is a potent inhibitor of the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis and the site at which exogenous cholesterol suppresses endogenous cholesterol synthesis. Because cholesterol sulfate inhibited sterologenesis in steroid-sulfatase deficient fibroblasts derived from patients with recessive X-linked ichthyosis, it was inferred that cholesterol sulfate per se and not cholesterol liberated by intracellular desulfation was the inhibitor in these studies. Cholesterol sulfate may be an endogenous regulator of mammalian cholesterol biosynthesis.     

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.     

Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.

The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown.     

Cholesterol inhibits glutamine metabolism in LLC WRC256 tumour cells but does not affect it in lymphocytes: possible implications for tumour cell proliferation

The effect of cholesterol on proliferation and glutamine metabolism of lymphocytes and tumour cells was investigated. The addition of cholesterol to the culture medium did not cause a significant effect on [2-(14)C]-thymidine incorporation in lymphocytes. In the presence of concanavalin A, lymphocyte proliferation was increased by cholesterol (from 0.013 up to 1.3 microm). At high concentrations (234 and 468 microm), however, a marked inhibition of lymphocyte proliferation occurred. The same inhibitory effect was observed in the presence of lipopolysaccharides. Cholesterol also caused a marked decrease of LLC WRC256 tumour cell growth at 117 and 234 microm. The same findings were obtained by the measurement of [2-(14)C]-thymidine incorporation in these cells. The effect of cholesterol on phosphate-dependent glutaminase activity was then tested in cultured lymphocytes and LLC WRC256 tumour cells. Cholesterol at concentrations of 117 and 234 microm did not alter this enzyme activity in lymphocytes. However, this sterol, already at 26 microm, caused a 44 per cent reduction in glutaminase activity. Similar to the changes observed for glutaminase, cholesterol reduced glutamine oxidation in LLC WRC256 tumour cells, whereas no effect was observed on lymphocytes. Therefore, cholesterol might control lymphocyte and tumour cells proliferation by different mechanisms. The significance of these findings for the immune function in tumour-bearing patients remains to be investigated.     

Differences in cholesterol transport by normal and malignant human kidney cells

Cholesterol uptake and release was measured in normal and malignant human kidney cells in culture. The time course and level of cholesterol uptake was the same in both normal and malignant cells and there was no accumulation of cholesterol. However, the time course of cholesterol release was strikingly different in normal $\text{versus}$ malignant cells. Release of cholesterol by normal cells plateaued early; whereas release by malignant cells continued at a linear rate. The results suggest that malignant cells lack either a competent mechanism for regulation of cholesterol synthesis or lack a mechanism for regulation of cholesterol efflux, thus leading to deregulation of cholesterol synthesis.     

Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression

Cholesterol is a major lipid component of the plasma membrane in animal cells. In addition to its structural requirement, cholesterol is essential in cell proliferation and other cell processes. The aim of the present study was to elucidate the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression, compared with other sterols of the cholesterol biosynthesis pathway. Human promyelocytic HL-60 cells were cultured in cholesterol-free medium and treated with different distal inhibitors of cholesterol biosynthesis (zaragozic acid, SKF 104976, SR 31747, BM 15766, and AY 9944), which allow the synthesis of isoprenoid derivatives and different sets of sterol intermediates, but not cholesterol. The results showed that only the inhibition of sterol Delta7-reductase was compatible with cell proliferation. Blocking cholesterol biosynthesis upstream of this enzyme resulted in the inhibition of cell proliferation and cell cycle arrest selectively in G2/M phase.     

Assembly of actin filaments induced by sequestration of membrane cholesterol in transformed cells

Cholesterol is a major lipid component of the plasma membrane that plays an important role in various signaling processes in mammalian cells. Our study is focused on the role of membrane cholesterol in the organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by a poorly developed actin network and less prominent stress fibers: human embryonic kidney HEK293, human epidermoid larynx carcinoma HEp-2, and mouse fibroblasts 3T3-SV40. Using Factin labeling with rhodamine phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin and polyene macrolide antibiotic filipin. The cells treated with these agents displayed similar reorganization of actin cytoskeleton involving filament assembly. In HEp-2 carcinoma cells and 3T3-SV40 fibroblasts, cholesterol-sequestering reagents induced intense stress fiber formation and enhanced cell spreading; i.e., features of transformed phenotype reversion were observed. The cytoskeleton rearrangements are probably initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.     

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.     

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints

Background

Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors.

Results

We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally.

Conclusion

In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.     

Regulation of sterol transport in human microvascular endothelial cells

In cultured human dermal microvessel endothelial cells, the rate of efflux (about twofold greater than for fibroblasts under equivalent conditions) was coupled to an equivalent high rate of sterol net transport from the cells to the medium. This net transport was linked with esterification via lecithin:cholesterol acyltransferase. Since the use of free sterol by plasma transferase is constant, such increased net transport indicates that endothelial cells are highly efficient, in competition with plasma lipoproteins, in supplying free sterol for esterification. These results indicate the marked ability of endothelial cells to regulate and maintain their sterol balance in the face of high sterol levels to which these cells are uniquely exposed in human plasma.     

Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts

Effects of cholesterol sulfate on acetate incorporation into lipid fractions were examined in normal human fibroblast and keratinocyte cultures. Inhibition of sterologenesis in normal fibroblast cultures by cholesterol sulfate was less profound than that produced by either lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate also inhibited sterologenesis in low density lipoprotein receptor-deficient fibroblasts and inhibited both sterologenesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in keratinocytes. Cholesterol sulfate increased incorporation of acetate into fatty acid-containing lipids in preconfluent cultures of both cell types in lipoprotein-depleted media. Similar effects were not observed either in response to lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate had no effect on oleic acid incorporation into diglycerides, triglycerides, or phospholipid fractions; neither did it inhibit acid lipase activity; nor did it inhibit fatty acid oxidation, indicating that cholesterol sulfate does not inhibit catabolism of acyl lipids. Because cholesterol sulfate had similar effects on fatty acid metabolism in steroid sulfatase-deficient fibroblasts lines, desulfation to cholesterol is not a prerequisite. Cholesterol sulfate did not significantly affect incorporation of oleic acid into sterol esters in fibroblast cultures, but in contrast, inhibited sterol esterification in keratinocyte cultures. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis.     

Scrambling of phospholipids activates red cell membrane cholesterol

Cholesterol is predicted to associate more strongly with the outer than the inner leaflet of plasma membrane bilayers based on the relative in vitro affinities of their phospholipids. Complex formation with the high-affinity species (especially saturated sphingomyelins) is said to reduce the chemical activity (escape potential or fugacity) of the sterol. We therefore tested the hypothesis that scrambling the sidedness of plasma membrane phospholipids of intact cells will increase the chemical activity of outer surface cholesterol. Upon activating the plasma membrane scramblase in intact human red cells by introducing ionomycin to raise cytoplasmic Ca++, phosphatidylserine became exposed and, concomitantly, the chemical activity of exofacial cholesterol was increased. (This was gauged by its susceptibility to cholesterol oxidase and its rate of transfer to cyclodextrin.) Similar behavior was observed in human fibroblasts. Two other treatments known to activate cell surface cholesterol (namely, exposure to glutaraldehyde and to low-ionic-strength buffer) also brought phosphatidylserine to the cell surface but by a Ca++-independent mechanism. Given that phospholipid scrambling is important in blood coagulation and apoptosis, the concomitant activation of cell surface cholesterol could contribute to these and other pathophysiological signaling processes.     

The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects.

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.