Effect Of 25-hydroxycholesterol Research Articles

Effect of 25-hydroxycholesterol in viral membrane fusion: Insights on HIV inhibition

Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection.Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane.With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC.

Effect of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol, on protein content, protein synthesis and LDH leakage in mouse epidermal cells

The cytotoxicity of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol was investigated in murine epidermal cell line, HEL-30. Lactate dehydrogenase (LDH) leakage, protein synthesis and protein content were determined after exposure of cell monolayers to the compounds ranging from 0.1–200 μM for 2, 6 or 24 h. 26-Aminocholesterol affected all the parameters studied time and concentration dependently; 25-hydroxycholesterol and 26-hydroxycholesterol were not toxic for HEL-30 cells. The cellular target for 26-aminocholesterol was primarily the membrane, since the LDH leakage was already detectable 10 min after exposure. On the other hand, for the other two oxysterols a protective role on this structure might be postulated. In fact 25-hydroxycholesterol and 26-hydroxycholesterol decreased the natural LDH leakage due to the ageing of the culture. In addition, 20 μM 25-hydroxycholesterol reversed the effect of moderately lytic doses of 26-aminocholesterol and Triton X-100, but not of sodium dodecyl sulfate.

The influence of 25-hydroxycholesterol on phosphatidylinositol content in cultured smooth muscle cells.

The effect of 25-hydroxycholesterol on phosphatidylinositol content was studied in subconfluent human arterial smooth muscle cells exposed to myo-[2-3H]inositol for 90 min and then to 25-hydroxycholesterol at different concentrations or for different incubating periods. Ethanol at a concentration of 0.05% in culture medium was used as control. The results demonstrate that an increased incubation time or an increase in the concentration of 25-hydroxycholesterol produced an increase in phosphatidylinositol and its degraded products. The ratio of precursor to product at each stage of phosphatidylinositol turnover in 25-hydroxycholesterol-treated cells, however, was not different from that in control cells. The data indicate that the presence of 25-hydroxycholesterol in the media induced general activation of phosphatidylinositol synthesis and turnover.

Oxysterols Inhibit Gap Junctional Communication between Rat Hepatocytes in Primary Culture

Several oxysterols were examined for their effect on gap junctional communication between rat hepatocytes in primary culture. 25-Hydroxycholesterol, 22(S)-hydroxycholesterol and 7 beta-hydroxycholesterol, in decreasing order of potency, markedly inhibited gap junctional communication. In contrast, 7-ketocholesterol showed no inhibitory effect. The inhibition of gap junctional communication by oxysterols was not a consequence of changes in cell viability, as measured by lactate dehydrogenase leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity. The addition of exogenous cholesterol to the culture medium did not abolish the effect of 25-hydroxycholesterol, suggesting that the capacity of oxysterols to inhibit gap junctional communication is independent of their inhibitory effect on cholesterol synthesis. We suppose that inhibition of gap junctional communication may be an early sign of oxysterols-induced toxicity on hepatocytes.

The effect of 25-hydroxycholesterol on the regulation of apolipoprotein E mRNA levels and secretion in the human hepatoma HepG2

The human hepatoma cell line, HepG2, was cultured with 25 OH cholesterol, a potent inhibitor of HMG-CoA reductase, in order to examine the effect of the oxysterol on apo E synthesis and secretion. Treatment of cells with oxysterol (2.5 μM) resulted in a greater than 90% inhibition of HMG-CoA reductase activity and a 3-fold reduction in its cognate mRNA level. However, apo E mRNA level and secretion were not affected after 24 h of drug treatment. This drug treatment was associated with a reduction in both cellular free and esterified cholesterol levels by 50% and 40%, respectively. Exposure of HepG2 cells to an ACAT inhibitor, the Sandoz compound (58-035) for 24 h, at a concentration of 5 μg/ml, resulted in a 30% increase and 70% decrease in the intracellular levels of free and esterified cholesterol, respectively. Under this regimen of drug treatment, the level of apo E mRNA was increased by approximately 70%, while HMG-CoA reductase mRNA level was decreased by 35%. When the cells were exposed to the combination of the ACAT inhibitor and 25 OH cholesterol, the cellular levels of free and esterified cholesterol were reduced by 30% and 80%, respectively. This combination of drugs had no effect on apo E mRNA; however, the level of HMG-CoA reductase mRNA was decreased by 3.5-fold. Taken together, the data suggested that reduction in the intracellular levels of either free or esterified cholesterol had no effect on apo E mRNA level. By contrast, a small increment in cellular free cholesterol content was associated with a significant induction in apo E mRNA level. Furthermore, 25 OH cholesterol caused a significant redistribution (50%) of apo E from the HDL fraction to the d > 1.21 g/ml infranatant. By using high performance liquid chromatography and molecular sieve columns, it was found that the appearance of a lipid-poor apo E particle was not an artifact of ultracentrifugation. This particle contained 85 wt% protein and 15 wt% of free cholesterol and phospholipid. The results suggested that a lipid-poor apo E particle was secreted by the HepG2 cells under certain circumstances.

HMG CoA reductase inhibitors affect Na(+)-H+ antiport activity in human lymphoblasts.

The Na(+)-H+ antiport is a membrane-bound glycoprotein that extrudes intracellular acid loads and regulates cellular volume. Cellular synthesis of the oligosaccharide side chains of glycoproteins is dependent on a supply of mevalonate, itself a product of the rate-limiting enzyme of cholesterol synthesis 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The effect of two HMG CoA reductase inhibitors (simvastatin and 25-hydroxycholesterol) on intracellular pH and Na(+)-H+ exchange was therefore studied. Inhibition of the Na(+)-H+ antiport by these agents led to a fall in intracellular pH but did not impair the regulatory volume increase response to a hypertonic stimulus. The inhibitory effect of simvastatin was prevented by mevalonate but not dolichol or squalene. The effect of 25-hydroxycholesterol was more complex and not easily reversed. Thus HMG CoA reductase inhibitors reduced the ability of human lymphoblasts to expel an intracellular acid load via the Na(+)-H+ antiport, although the response of the antiport to an osmotic stimulus was preserved.

The effect of 25-hydroxycholesterol on accumulation of intracellular calcium

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37°C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca 2++Mg 2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca 2++Mg 2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca 2++Mg 2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.

Regulation of acyl CoA:cholesterol acyltransferase by 25-hydroxycholesterol in rabbit intestinal microsomes and absorptive cells.

The regulation of rabbit intestinal acyl CoA:cholesterol acyltransferase (ACAT) by 25-hydroxycholesterol was studied. 25-Hydroxycholesterol significantly increased jejunal microsomal ACAT activity. The stimulation of ACAT activity by 25-hydroxycholesterol was inversely related to microsomal cholesterol content. In enterocytes, 25-hydroxycholesterol stimulated cholesteryl ester synthesis 6-fold. The esterification of cholesterol, taken up by enterocytes from liposomes or bile salt micelles, was increased by 25-hydroxycholesterol. This, however, did not affect the rate of uptake of cholesterol by the cells. In intestinal cells from rabbits fed cholesterol, the effect of 25-hydroxycholesterol on cholesteryl ester synthesis was 50% of that in cells prepared from animals fed no cholesterol. 25-Hydroxycholesterol stimulated the esterification of newly synthesized cholesterol. As new free cholesterol increased in enterocytes, 25-hydroxycholesterol lost its effect. Despite large amounts of newly synthesized cholesterol, oleic acid incorporation into cholesteryl ester was not increased. We conclude that 25-hydroxycholesterol increases intestinal ACAT activity. The effect of 25-hydroxycholesterol on ACAT is dependent upon the availability of cholesterol to the enzyme. At cholesterol concentrations below saturation, the oxygenated sterol has a stimulatory effect. If ACAT is saturated, 25-hydroxycholesterol has no effect. ACAT catalyzes the esterification of two separate pools of cholesterol within the enterocyte, i.e., newly synthesized cholesterol and membrane cholesterol. 25-Hydroxycholesterol increases the esterification rate of cholesterol in both pools.

Effect of 25-hydroxycholesterol and dexamethasone on sterol synthesis in isolated mouse thymocytes

Oxygenated derivatives of cholesterol, such as 25-OH-cholesterol, are potent inhibitors of cholesterol synthesis in various cell types. Although the main site of 25-OH-cholesterol action appears to be mediated by the inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, one of the key steps in the pathway of cholesterol formation, there is no indication on the molecular mechanism leading to enzyme inhibition. We have therefore compared in isolated mouse thymocytes the mode of action of 25-OH-cholesterol to that of dexamethasone which has also been demonstrated to block cholesterol synthesis. 25-OH-cholesterol induced a rapid, dose dependent, inhibition of [ 3H]acetate incorporation into the sterol fraction. This effect was specific for the cholesterol biosynthetic pathway and, in contrast to dexamethasone, did not affect either lipid synthesis or RNA formation. Unlike glucocorticoid action, the effect of 25-OH-cholesterol was not suppressed in the presence of actinomycin D and did not appear to be mediated via an interaction with specific receptors. It thus appears that the mechanism of action of 25-OH-cholesterol on cholesterol biosynthesis is not comparable to that of steroid hormones.

Radioimmune precipitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Chinese hamster fibroblasts. Effect of 25-hydroxycholesterol.

Antibody prepared against 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of rat liver can be shown to inhibit this enzyme in extracts prepared from cultured Chinese hamster ovary (CHO-K1) cells. The molecular weight (53,000) of the HMG-CoA reductase subunits of rat liver and Chinese hamster liver is identical with a [35S]methionine-labeled polypeptide that can be precipitated from CHO-K1 lysates by this antibody used in conjunction with protein A Sepharose. It is shown that 25-hydroxycholesterol which lowers HMG-CoA reductase activity in cultured fibroblasts blocks the incorporation of labeled methionine into this polypeptide. Furthermore, the antibody immune precipitates two other polypeptides with molecular weights of 127,000 and 60,000. The latter polypeptide responds to 25-hydroxycholesterol in the same fashion as the 53,000-dalton polypeptide. In a dominant 25-hydroxycholesterol-resistant mutant of the CHO-K1 cell, 25-hydroxycholesterol did not inhibit incorporation of labeled methionine into either the 53,000- or 60,000-dalton polypeptides.

Effects of 25-hydroxycholesterol and aminoglutethimide in isolated rat adrenal cells. A model for congenital lipoid adrenal hyperplasia?

The production of corticosterone from 25-hydroxycholesterol by isolated rat adrenal cells is inhibited by aminoglutethimide phosphate (AGI); half-maximal inhibition is obtained at ca. 10 μM. AGI also inhibits ACTH-stimulated steroid production from endogeneous substrates; here half-maximal inhibition is obtained with ca. 40 μM AGI. In the presence of ACTH + AGI, 25-hydroxycholesterol causes additive inhibition. This effect of 25-hydroxycholesterol is dose-dependent. ACTH-stimulated steroid production from endogeneous substrates is partially inhibited by 5-cholene-3β, 24-diol. These results may just reflect substrate competition for the side-chain cleaving system or may be due to some secondary toxic effect on the cells.