Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level.
The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.
Choi
J W
JW
University of Health Sciences, Chicago Medical School, Department of Pharmacology and Molecular Biology, IL 60064.
Lundquist
E N
EN
Peffley
D M
DM
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology*; D003609:Dactinomycin / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006888:Hydroxycholesterols / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D014158:Transcription, Genetic
D006888:Hydroxycholesterols; D012333:RNA, Messenger; D003609:Dactinomycin; C007997:25-hydroxycholesterol; D003513:Cycloheximide; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases