monensin and 25-hydroxycholesterol

Expression of the lipophilic ligand transporter, apolipoprotein D (apoD) by primary astrocyte cultures derived from neonatal mouse brain was investigated. Western blot analysis of cell lysates and media showed that apoD is constitutively secreted by astrocytes with little intracellular storage. The secreted apoD floated primarily at density 1.063-1.21 g ml-1 upon sequential ultracentrifugation indicating its association with lipids. Treatment of astrocytes with the carboxylic ionophore, monensin, resulted in intracellular retention and decreased secretion of apoD that was of slightly reduced M(r). Progesterone, a steroid hormone that binds to apoD with high affinity (10(-6) mol l-1) and the oxysterol, 25-hydroxycholesterol which is a potent regulator of cellular cholesterol homeostasis in mammalian cells, differentially stimulated apoD, but not apoE secretion. These results show that astrocytes synthesize and constitutively secrete apoD and suggest a physiologic role for this lipocalin in cholesterol metabolism in the nervous system.

Topics: Animals; Animals, Newborn; Apolipoproteins; Apolipoproteins D; Apolipoproteins E; Astrocytes; Biological Transport; Cells, Cultured; Hydroxycholesterols; Ligands; Lipid Metabolism; Mice; Monensin; Progesterone; Solubility

The effect of disruption of the Golgi apparatus on 25-hydroxycholesterol-mediated transcriptional suppression and activation of acyl-CoA:cholesterol acyltransferase was examined. In Chinese hamster ovary (CHO) cells, brefeldin A (BFA) caused dose-dependent inhibition of 25-hydroxycholesterol-mediated suppression of mRNAs for four sterol-regulated genes: 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase, and the low density lipoprotein receptor. BFA prevented suppression whether added prior to or following a 4-h pretreatment with 25-hydroxycholesterol. In the presence of BFA (1 microgram/ml), 25-hydroxycholesterol-mediated suppression of mRNAs for HMG-CoA reductase, the low density lipoprotein receptor, and farnesyl-diphosphate synthase was almost completely blocked. HMG-CoA synthase mRNA was 80-90% suppressed by 25-hydroxycholesterol compared with 50-60% suppression in the presence of BFA. These effects of BFA were not due to alterations in mRNA stability. Disruption of the Golgi apparatus, as assessed by staining with a fluorescent lectin, correlated with concentrations of BFA that reversed mRNA suppression. Monensin was also found to block the effects of 25-hydroxycholesterol on suppression of HMG-CoA reductase. However, this ionophore decreased the other three sterol-regulated mRNAs to a similar degree as 25-hydroxycholesterol. In contrast to CHO cells, BFA-resistant PtK1 cells displayed normal down-regulation of HMG-CoA reductase and an intact Golgi apparatus in the presence of BFA and 25-hydroxycholesterol. Cholesterol esterification in CHO cells was stimulated to a similar extent by BFA (1 microgram/ml) and 25-hydroxycholesterol, and simultaneous treatment of CHO cells with both compounds was 60-70% additive. These results suggest that an intact Golgi apparatus is required for 25-hydroxycholesterol-mediated suppression of mRNA.

Topics: Animals; Brefeldin A; Cell Line; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Cyclopentanes; Dose-Response Relationship, Drug; Esterification; Gene Expression Regulation; Golgi Apparatus; Hydroxycholesterols; Marsupialia; Monensin; Protein Synthesis Inhibitors; RNA, Messenger; Transcription, Genetic

A monensin-resistant clone (Monr-31) shows a related series of differences from its parental Chinese hamster ovary (CHO) cell line in the cellular response to several ligands. The uptake and metabolism of low density lipoprotein (LDL) in the mutant cells are defective. Accumulation of fluorescent-labeled LDL as well as internalization and degradation of 125I-LDL are greatly reduced in Monr-31 cells. The receptor number for LDL on the cell surface of Monr-31 is about one-third that for CHO cells, but affinity constants for both cell lines are similar. Electrophoretic analysis shows a slightly reduced molecular weight of LDL receptor in Monr-31 cells in comparison to that in CHO cells. The internalization index (internalization plus degradation per binding) of LDL of the mutant is about one-half that of CHO cells, suggesting a failure of internalization of LDL as well as LDL binding. Hybrids (hyb-1, -2, and -3) between CHO and Monr-31 cells show LDL binding and LDL internalization activities comparable to that of CHO cells, suggesting that the altered LDL response in Monr-31 cells is recessive. Addition of exogenous LDL to culture medium down-regulates the LDL receptor activity of CHO, hyb-2, and hyb-3 cells, whereas no such down-regulation is seen in Monr-31 cells. Probably as a result of the failure of down-regulation, the prominent inhibition of sterol synthesis from acetate and 3-hydroxy-3-methylglutaryl-coenzyme A reductase observed in CHO cells is scarcely detectable in Monr-31 cells. As a correlated result, sterol synthesis from acetate is 6-fold higher in the mutant. The failure of down-regulation of LDL receptors in Monr-31 cells is discussed in relation to the altered binding and internalization of LDL.

Topics: Animals; Cell Line; Cholesterol; Cricetinae; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Female; Hybrid Cells; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins, LDL; Molecular Weight; Monensin; Mutation; Ovary; Receptors, LDL; Sterols