Choi J W, Lundquist E N, Peffley D M
University of Health Sciences, Chicago Medical School, Department of Pharmacology and Molecular Biology, IL 60064.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):859-66. doi: 10.1042/bj2960859.
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.
在幼仓鼠肾细胞系C100中评估了蛋白质合成抑制剂放线菌酮对25-羟基胆固醇介导的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶mRNA水平抑制作用的影响。在补充了脱脂胎牛血清和25μM洛伐他汀的培养基中培养12 - 24小时的细胞，其HMG-CoA还原酶mRNA水平比在补充了未脱脂胎牛血清（FBS）的培养基中生长的细胞高5倍。如通过分离细胞核的连续转录分析所确定，较高水平是由于转录增加所致。向用洛伐他汀处理的细胞中添加25-羟基胆固醇，在处理后4小时内将HMG-CoA还原酶mRNA水平降至在补充FBS培养基中生长的细胞的水平。这种降低部分是由于基因转录减少。与25-羟基胆固醇一起添加到用洛伐他汀处理的细胞中的放线菌酮阻断了mRNA水平的抑制，但没有阻断氧甾醇介导的转录抑制。此外，添加到在补充FBS培养基中生长的细胞中的放线菌酮相对于未处理的细胞迅速将mRNA水平提高了10倍，而转录没有可比的增加。在这种条件下，未观察到β-肌动蛋白的mRNA水平或转录速率有可比的增加。这些结果表明，在氧甾醇存在下，放线菌酮特异性地稳定HMG-CoA还原酶mRNA，并表明氧甾醇在转录后水平介导的HMG-CoA还原酶mRNA抑制需要持续合成一种寿命较短的蛋白质调节因子。
