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

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 μg/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 nRNA was 80-90% suppressed by 25-hydroxy- cholesterol 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-hydroxy-cholesterol. Cholesterol esterification in CHO cells was stimulated to a similar extent by BFA (1 μg/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.