Brefeldin A renders Chinese hamster ovary cells insensitive to transcriptional suppression by 25-hydroxycholesterol.
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.
Ridgway
N D
ND
Department of Pediatrics and Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Lagace
T A
TA
D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003517:Cyclopentanes / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004951:Esterification; D005786:Gene Expression Regulation / Q000187:drug effects; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008394:Marsupialia; D008985:Monensin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D003517:Cyclopentanes; D006888:Hydroxycholesterols; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D020126:Brefeldin A; C007997:25-hydroxycholesterol; D008985:Monensin; D002784:Cholesterol