Brefeldin A renders Chinese hamster ovary cells insensitive to transcriptional suppression by 25-hydroxycholesterol

J Biol Chem. 1995 Apr 7;270(14):8023-31. doi: 10.1074/jbc.270.14.8023.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brefeldin A
  • CHO Cells
  • Cell Line
  • Cholesterol / metabolism
  • Cricetinae
  • Cricetulus
  • Cyclopentanes / pharmacology*
  • Dose-Response Relationship, Drug
  • Esterification
  • Gene Expression Regulation / drug effects
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism*
  • Hydroxycholesterols / antagonists & inhibitors
  • Hydroxycholesterols / pharmacology*
  • Marsupialia
  • Monensin / pharmacology
  • Protein Synthesis Inhibitors / pharmacology
  • RNA, Messenger / metabolism
  • Transcription, Genetic / drug effects*

Substances

  • Cyclopentanes
  • Hydroxycholesterols
  • Protein Synthesis Inhibitors
  • RNA, Messenger
  • Brefeldin A
  • 25-hydroxycholesterol
  • Monensin
  • Cholesterol