Wilkin D J, Kutsunai S Y, Edwards P A
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1679.
J Biol Chem. 1990 Mar 15;265(8):4607-14.
We report the isolation and nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA, an enzyme in the cholesterogenic pathway. Partial cDNAs for the human farnesyl pyrophosphate synthetase were isolated by screening human hepatoma (HepG2) and placental cDNA libraries with the rat liver cDNA for farnesyl pyrophosphate synthetase as a probe. Anchored polymerase chain reaction was used to isolate the 5'-end of the cDNA. The nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA has high identity (86%) to the rat liver cDNA. Treatment of the human monocytic leukemia cell line THP-1 with phorbol esters led to 2--7-fold increases in mRNA concentrations for the three cholesterogenic enzymes, farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and HMG-CoA synthase within 5 h. Immunoprecipitation of radiolabeled cells demonstrated that there was a corresponding increase in the rate of synthesis of all three proteins. The addition of cycloheximide to cells also led to increases in the mRNA concentrations of the three enzymes. Treatment of cells with phorbol esters and cycloheximide resulted in superinduction of all three mRNAs; HMG-CoA synthase mRNA levels increased 35-fold, farnesyl pyrophosphate synthetase 17-fold, and HMG-CoA reductase 16-fold 5 h after treatment. The mRNA levels returned to pretreatment levels by 20 h. Cells were also preincubated in the presence of a lipoprotein-deficient fraction of serum plus mevinolin to induce the levels of the three mRNAs. Addition of phorbol esters and cycloheximide to these derepressed cells led to further increases in the mRNA levels for all three enzymes. These results are consistent with the hypothesis that THP-1 cells contain a short-lived negative transcription factor which regulates transcription of the FPP synthetase, HMG-CoA reductase, and HMG-CoA synthase genes. Phorbol esters also regulate these same genes, presumably by modifying a common negative transcription factor and/or by inducing a positive transcription factor(s).
我们报道了人法尼基焦磷酸合成酶cDNA的分离及核苷酸序列，该酶是胆固醇生成途径中的一种酶。以大鼠肝脏法尼基焦磷酸合成酶的cDNA为探针，通过筛选人肝癌（HepG2）和胎盘cDNA文库，分离出了人法尼基焦磷酸合成酶的部分cDNA。采用锚定聚合酶链反应分离cDNA的5′端。人法尼基焦磷酸合成酶cDNA的核苷酸序列与大鼠肝脏cDNA具有高度同源性（86%）。用佛波酯处理人单核细胞白血病细胞系THP-1，在5小时内，三种胆固醇生成酶，即法尼基焦磷酸合成酶、3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶和HMG-CoA合酶的mRNA浓度增加了2至7倍。对放射性标记细胞进行免疫沉淀表明，这三种蛋白质的合成速率相应增加。向细胞中添加放线菌酮也导致这三种酶的mRNA浓度增加。用佛波酯和放线菌酮处理细胞导致所有三种mRNA的超诱导；处理5小时后，HMG-CoA合酶mRNA水平增加35倍，法尼基焦磷酸合成酶增加17倍，HMG-CoA还原酶增加16倍。20小时后，mRNA水平恢复到预处理水平。细胞还在缺乏脂蛋白的血清部分加美伐他汀存在下预孵育，以诱导这三种mRNA的水平。向这些去阻遏细胞中添加佛波酯和放线菌酮导致所有三种酶的mRNA水平进一步增加。这些结果与以下假设一致，即THP-1细胞含有一种寿命较短的负转录因子，该因子调节FPP合成酶、HMG-CoA还原酶和HMG-CoA合酶基因的转录。佛波酯也调节这些相同的基因，可能是通过修饰一个共同的负转录因子和/或诱导一个正转录因子。
