Lipid-deficient Serum Research Articles

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

The Lamin B Receptor Provides Sterol Reductase Activities Critical to Neutrophil Development: Implications for Cholesterol Biosynthesis In Regulating Myelopoiesis

AbstractAbstract 389The lamin B receptor (LBR) is a unique nuclear envelope protein whose expression is critical to the correct morphologic maturation of differentiating neutrophils. Heterozygous mutations of LBR in humans cause Pelger-Huët anomaly (PHA), a disorder characterized by hypolobulated neutrophil nuclei. Homozygous mutations of LBR cause the lethal disorder HEM/Greenberg skeletal dysplasia in humans and the ichthyosis (ic) phenotype in mice, both of which are associated with severe skeletal defects and severely hypolobulated, or mononuclear neutrophils. Why complete loss of LBR expression causes neutrophil hypolobulation and severe developmental defects remains unclear. The N-terminal domain of LBR, which resides in the nucleoplasm, binds to B-type lamins and chromatin, which is thought to facilitate nuclear envelope reformation at the end of mitosis. The C-terminal domain contains 8 membrane-spanning segments and is homologous to the sterol Δ14 reductase TM7SF2. Thus phenotypes caused by loss of LBR expression have been considered as laminopathies, i.e. due to disruptions to the functions of lamin B, or due to a disruption in cholesterol biosynthesis. Cholesterol plays a vital role in the production of lipid-enriched microdomains termed lipid rafts, which facilitate the assembly of membrane proteins important for neutrophil adhesion, chemotaxis and the respiratory burst. Furthermore, lipid depravation inhibits the proliferation of myeloblastic HL-60 cells, indicating that cholesterol levels are important to myeloid cell growth. We generated a novel EML progenitor line from the bone marrow of a homozygous ic mouse, termed EML-ic/ic cells, which can be induced to promyelocytic EPRO-ic/ic cells and then into mature neutrophils that display severe PHA identical to those found in ichthyosis mice. To further investigate the importance of cholesterol biosynthesis to neutrophil growth and maturation, we analyzed wild-type EML cells for growth defects when cultured in lipid-deficient serum (LPDS) or when treated with molecules that either deplete cellular cholesterol levels (lovastatin) or sequester membrane cholesterol (methyl-β-cyclodextrin, MβCD). Growth of EML cells was modestly inhibited in all three conditions, but growth of EPRO cells was severely affected. Lipid levels produced by the cells increased as differentiation progressed, suggesting that myeloid progenitors exhibit increased demands for cholesterol as they mature into terminally differentiated neutrophils. By comparison, growth of EML- or EPRO-ic/ic cells was dramatically inhibited when cultured in LPDS, lovastatin or MβCD. Lipid production also was compromised in the ic/ic cells at all stages of development. Both of these defects displayed by EML-ic/ic cells were significantly improved by ectopic expression of a wild-type Lbr. Expression of mutant forms of Lbr that contained the C-terminal domain homologous to TM7SF2, but that lacked binding domains for lamin B or chromatin, also improved proliferation in cholesterol depleted conditions and increased lipid production. Interestingly, while expression of the wild-type Lbr restored nuclear lobulation to the vast majority of ic/ic neutrophils, the mutant forms also were able to rescue lobulation in numerous ic/ic cells. The previously identified respiratory burst defect produced by ic/ic neutrophils also was partly reversed by both wild-type and mutant forms of Lbr. Finally, a Northern analysis demonstrated that neutrophil differentiation caused increased Lbr expression whereas Tm7sf2 expression did not change. In contrast, sterol depravation activated Tm7sf2 expression but did not affect Lbr expression. Together these data indicate that cholesterol biosynthesis is important to the growth and function of neutrophils, that demands for cholesterol increase as neutrophils mature, and that increased Lbr expression during neutrophil differentiation functions to meet these demands. In addition, C-terminal domains of Lbr can support the roles of Lbr in mediating neutrophil survival, morphologic maturation and functional responses. Our data provide new insight into the complex roles that cholesterol play in myelopoiesis, and may be important to understanding the effects of dysfunctional cholesterol biosynthesis to innate immunity provided by neutrophils. Disclosures:No relevant conflicts of interest to declare.

25-hydroxycholesterol acts in the Golgi compartment to induce degradation of tyrosinase.

Oxysterols play a significant role in cholesterol homeostasis. 25-Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol-binding protein and oxysterol-related proteins, the sterol regulatory element binding protein, and the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC(50) of 0.34 microM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse-chase studies of 25HC-treated cells demonstrated enhanced degradation of tyrosinase, the rate-limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis-Golgi SNARE protein complex, were also diminished in 25HC-treated melanocytes, however levels of the ER chaperone calnexin and the cis-Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4 alpha-allylcholestan-3 alpha-ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.

Internalization of Transthyretin: EVIDENCE OF A NOVEL YET UNIDENTIFIED RECEPTOR-ASSOCIATED PROTEIN (RAP)-SENSITIVE RECEPTOR

Transthyretin (TTR) is a plasma carrier of thyroxine and retinol-binding protein (RBP). Though the liver is the major site of TTR degradation, its cellular uptake is poorly understood. We explored TTR uptake using hepatomas and primary hepatocytes and showed internalization by a specific receptor. RBP complexed with TTR led to a 70% decrease of TTR internalization, whereas TTR bound to thyroxine led to a 20% increase. Different TTR mutants showed differences in uptake, suggesting receptor recognition dependent on the structure of TTR. Cross-linking studies using hepatomas and (125)I-TTR revealed a approximately 90-kDa complex corresponding to (125)I-TTR bound to its receptor. Given previous evidence that a fraction of TTR is associated with high-density lipoproteins (HDL) and that in the kidney, megalin, a member of the low-density lipoprotein receptor family (LDLr) internalizes TTR, we hypothesized that TTR and lipoproteins could share related degradation pathways. Using lipid-deficient serum in uptake assays, no significant changes were observed showing that TTR uptake is not lipoprotein-dependent or due to TTR-lipoprotein complexes. However, competition studies showed that lipoproteins inhibit TTR internalization. The scavenger receptor SR-BI, a HDL receptor, and known LDLr family hepatic receptors did not mediate TTR uptake as assessed using different cellular systems. Interestingly, the receptor-associated protein (RAP), a ligand for all members of the LDLr, was able to inhibit TTR internalization. Moreover, the approximately 90-kDa TTR-receptor complex obtained by cross-linking was sensitive to the presence of RAP. To confirm that RAP sensitivity observed in hepatomas did not represent a mechanism absent in normal cells, primary hepatocytes were tested, and similar results were obtained. The RAP-sensitive TTR internalization together with displacement of TTR uptake by lipoproteins, further suggests that a common pathway might exist between TTR and lipoprotein metabolism and that an as yet unidentified RAP-sensitive receptor mediates TTR uptake.

Lipoprotein-X fails to inhibit hydroxymethylglutaryl coenzyme A reductase in HepG2 cells

Abnormalities of lipid composition and metabolism are frequently observed in patients with cholestatic liver disease. Both elevated low-density lipoprotein (LDL) levels and the appearance of lipoprotein-X (LP-X) in plasma underlie the high incidence of hypercholesterolemia in this population. We tested the hypothesis that the hypercholesterolemia of cholestasis may reflect a failure of normal feedback regulation of hepatic cholesterolgenesis by determining the influence of LP-X on the rate-limiting enzyme of cholesterol synthesis, hydroxymethylglutaryl coenzyme A (HMG CoA) reductase. Cultured human hepatoma (HepG2) cells were incubated in purified lipoprotein for 24 hours, harvested, and then assayed for HMG CoA reductase activity and mass. LDL isolated from either normal controls or patients with cholestasis decreased reductase activity in a dose-dependent fashion (2 to 30 μg cholesterol/mL media) to a level approximately 50% of that measured in cells incubated in lipid-deficient serum. LP-X failed to downregulate enzyme activity compared with LDL, with little change in reductase activity at cholesterol concentrations (30 μg/mL media) that produced maximal reductase inhibition by LDL. Three distinct LP-X subspecies were purified from the plasma of a patient with primary biliary cirrhosis (PBC) and tested in an analogous manner. All LP-X subspecies were similar in their inability to decrease reductase activity as compared with LDL. HMG CoA reductase mass was increased approximately twofold in cells incubated with LP-X, as estimated by Western blot analysis. These results suggest that LP-X may contribute to hypercholesterolemia in the cholestatic patient by not effectively downregulating hepatic cholesterol synthesis.

Long term treatment of neonatal aortic smooth muscle cells with βVLDL induces cholesterol accumulation

A model for smooth muscle derived foam cells was developed by treating smooth muscle cells isolated from the aortae of neonatal rabbits with βVLDL for up to 1 month. Hyperlipidemic βVLDL isolated from cholesterol fed rabbits induced proliferation of the cells that were maintained in lipid deficient serum. In addition, the lipoprotein fraction stimulated [ 14C]oleic acid incorporation into [ 14C]cholesteryl ester, even in cultures that had been chronically exposed to the lipoprotein. The accumulation of cholesterol was evaluated and small amounts of cholesteryl ester were demonstrated in cultures treated for 3 days with βVLDL. However, continued exposure to the lipoprotein resulted in larger elevations in total cholesterol, approximately 65% of which was in the esterified form in cultures treated with 100 wg OVLDL/ml for 24 days. When cholesterol levels were examined as a function of time, it was determined that both total cholesterol and cholesteryl ester levels increased. Approximately 2–3 weeks after lipoprotein was introduced to the culture, maximum levels were attained. Triglyceride levels were also measured and found to increase more than two-fold in cultures that had been incubated in the presence of βVLDL for 24 days, when compared to cultures incubated in its absence. Examination of the cultures by electron microscopy revealed intracytoplasmic lipid droplets in βVLDL treated cells. These results suggest that βVLDL treatment of neonatal aortic smooth muscle cells provides an ideal model in which to study the lipid laden smooth muscle cells that characterize the atherosclerotic plaque.

Inhibitors of sterol synthesis. Studies of the metabolism of 5α-cholest-8(14)-en-3β-ol-15-one in Chinese hamster ovary cells and its effects on activities of early enzymes in cholesterol biosynthesis

The metabolism of [2,4- 3]5α-cholest-8(14)-en-3β-ol-15-one (I) has been studied in Chinese hamster ovary (CHO-K1) cells which were maintained in a lipid-deficient medium. The incorporaton of I into the cells was linear with respect to sterol concentration in the medium over the ranges of concentrations studied and was more than 3.5 times that of the uptake of cholesterol. The results of detailed chromatographic analyses of the lipids recovered from the cells after 6 h of incubation with [2,4- 3H]I (0.5 μM or 6.0 μM) indicated that most of the 3H was associated with free I. Considerably lesser amounts of the 3H was associated with esters of I. No formation of [ 3H]cholesterol or [ 3H]cholesteryl esters (or other C 27 monohydroxysterols) from labeled I was observed. The labeled material with the chromatographic behavior of the esters of I gave, after mild alkaline hydrolysis, the free 15-ketosterol which was characterized by the results of chromatographic and cocrystallization studies. Upon transfer of the CHO-K1 cells from a culture medium containing 8% newborn calf serum to the same medium containing 8% lipid-deficient newborn calf serum, increases in the levels of activity of cytosolic acetoacetyl-CoA thiolaseand 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and of HMG-CoA reductase were observed. These increases were blocked by the addition of I at a concentration of 1.0 μM. I (1.0 μM) also caused a decrease in the levels of activity of the three enzymes in cells previously grown in medium containing lipid-deficient serum. These results demonstrate that I not only affects the enzymatic reduction of HMG-CoA but also the enzymatic formation of this key intermediate in cholesterol biosynthesis.

Control of phosphorylation and decarboxylation of mevalonic acid and its metabolites in cultured human fibroblasts and in rat liver in vivo.

The activity of mevalonate kinase and of mevalonate pyrophosphate decarboxylase in human skin fibroblasts grown in culture was increased when whole fetal calf serum in the incubation medium was replaced with lipid-deficient serum. The drug demecolcine interfered with low density lipoprotein binding by cells and increased sterol synthesis and activities of hydroxymethylglutaryl-CoA reductase and mevalonate kinase. In contrast to normal cells, fibroblasts from a patient with homozygous familial hypercholesterolemia did not show any lower mevalonate kinase activity following incubation with whole serum compared with that in cells incubated with lipid-deficient serum. Insulin increased the activity of mevalonate kinase in fibroblasts. Livers of rats fed for 7 days with a diet containing 1% cholesterol showed reduced activity of mevalonate kinase and mevalonate phosphate kinase. These results are consistent with the possibility that enzymatic reactions other than those catalyzed by hydroxymethylglutaryl-CoA reductase may play a role in the physiological regulation of sterol synthesis in mammalian tissues.

Huntington's disease--growth of fibroblast cultures in lipid-deficient medium: a preliminary report.

When skin fibroblast cultures derived from subjects with Huntington's disease (HD) were grown in medium supplemented with lipid-deficient serum (LDS), growth rates were less than those of control cultures. The mean percentage increase in generation times for nine control cultures incurred by using LDS rather than fetal calf serum (FCS) was 8.0%. Four cultures from 2 patients with HD failed to grow in LDS but grew well in medium supplemented with FCS. In nine other cultures derived from 7 patients with HD the mean percentage increase in generation time incurred by using LDS was 88.1%. Returning cultures to FCS or adding a mixture of linoleic and linolenic acids restored the growth rate of HD cultures to normal.