Straka M S, Panini S R
Eleanor Roosevelt Institute, Denver, Colorado 80206.
Arch Biochem Biophys. 1995 Feb 20;317(1):235-43. doi: 10.1006/abbi.1995.1158.
We have examined the mechanisms of sterol-independent regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase by mevalonate in Chinese hamster ovary (CHO) cells. Serum lipoproteins, 25-hydroxycholesterol, or mevalonate each repress HMG-CoA reductase activity by fivefold or more, and mevalonate lowers the rate of reductase synthesis by twofold. However, while the expression of the HMG-CoA reductase promoter construct, T42 delta CAT, in stable transfectants is also repressed by serum lipoproteins and 25-hydroxycholesterol, mevalonate is without effect. In addition, while 25-hydroxycholesterol reduces the steady-state level of endogenous HMG-CoA reductase mRNA by more than threefold, mevalonate again has no effect. Mevalonate does partially regulate the expression of both the artificial promoter construct pTK-Kx3-CAT, containing three copies of the sterol regulatory element, SRE-1, and the full-length LDL receptor promoter construct, pLDLRCAT-6500 as well as the expression of functional LDL receptors. This transcriptional regulation appears to be mediated by sterol end products generated from added mevalonate. In CHO cells starved for mevalonate due to a mutation in the biosynthetic pathway, addition of 20 mM mevalonate accelerates the rate of degradation of HMG-CoA reductase by threefold whether new sterol biosynthesis is blocked or not. In such cells, addition of 25-hydroxycholesterol, by itself, also decreases the half-life of reductase from 11.6 to 2.3 h. In contrast, in cells acutely treated with a reductase inhibitor, sterol-accelerated degradation of reductase is only observed in the presence of submillimolar level of mevalonate. We conclude that large concentrations of exogenous mevalonate fail to generate a transcriptional regulator of HMG-CoA reductase in CHO cells but do lead to the formation of translational regulator(s) of reductase synthesis. In contrast, sterol regulators derived from exogenous mevalonate appear to be capable of downregulating the LDL receptor promoter. We further conclude that in the absence of pretreatment with a reductase inhibitor, the regulatory signals generated by sterols and nonsterols for accelerated degradation of HMG-CoA reductase are mutually independent. However, the enzyme synthesized in the presence of reductase inhibitors appears to exhibit an obligatory corequirement for low-dose mevalonate for sterol-accelerated degradation.
我们研究了甲羟戊酸对中国仓鼠卵巢（CHO）细胞中3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶表达的非甾醇依赖性调节机制。血清脂蛋白、25-羟基胆固醇或甲羟戊酸均可使HMG-CoA还原酶活性降低五倍或更多，且甲羟戊酸使还原酶的合成速率降低两倍。然而，虽然在稳定转染子中HMG-CoA还原酶启动子构建体T42δCAT的表达也受到血清脂蛋白和25-羟基胆固醇的抑制，但甲羟戊酸却没有作用。此外，虽然25-羟基胆固醇使内源性HMG-CoA还原酶mRNA的稳态水平降低了三倍多，但甲羟戊酸同样没有作用。甲羟戊酸确实部分调节了人工启动子构建体pTK-Kx3-CAT（含有三个甾醇调节元件SRE-1拷贝）和全长低密度脂蛋白（LDL）受体启动子构建体pLDLRCAT-6500的表达以及功能性LDL受体的表达。这种转录调节似乎是由添加的甲羟戊酸产生的甾醇终产物介导的。在由于生物合成途径突变而缺乏甲羟戊酸的CHO细胞中，无论新的甾醇生物合成是否被阻断，添加20 mM甲羟戊酸都会使HMG-CoA还原酶的降解速率加快三倍。在这类细胞中，单独添加25-羟基胆固醇也会使还原酶的半衰期从11.6小时降至2.3小时。相比之下，在用还原酶抑制剂急性处理的细胞中，只有在存在亚毫摩尔水平的甲羟戊酸时才会观察到甾醇加速的还原酶降解。我们得出结论，高浓度的外源性甲羟戊酸无法在CHO细胞中产生HMG-CoA还原酶的转录调节因子，但确实会导致还原酶合成的翻译调节因子的形成。相比之下，源自外源性甲羟戊酸的甾醇调节因子似乎能够下调LDL受体启动子。我们进一步得出结论，在没有用还原酶抑制剂预处理的情况下，甾醇和非甾醇产生的用于加速HMG-CoA还原酶降解的调节信号是相互独立的。然而，在还原酶抑制剂存在下合成的酶似乎对低剂量甲羟戊酸有强制性共同需求，以实现甾醇加速的降解。
