Sharkey M F, Miyanohara A, Elam R L, Friedmann T, Witztum J L
Department of Medicine and Pediatrics, University of California, San Diego, La Jolla 92093.
J Lipid Res. 1990 Dec;31(12):2167-78.
We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.
我们曾报道使用逆转录病毒载体将低密度脂蛋白（LDL）受体基因导入缺乏受体的渡边遗传性高脂血症（WHHL）兔成纤维细胞（宫原，A.，M.F.沙基，D.斯坦伯格，J.L.维茨图姆，和T.弗里德曼。1988年。美国国家科学院院刊。85: 6538 - 6542）。由于LDL受体的cDNA不包含赋予固醇介导的LDL受体转录抑制作用的5'固醇调节元件，我们预计该载体转导的LDL受体活性不会对固醇产生反应。然而，我们现在证明，在受感染的细胞中，固醇通过一种似乎在转录后水平介导的机制对受体蛋白进行下调。当受感染的WHHL细胞与LDL或胆固醇加25 - 羟胆固醇预孵育时，LDL受体活性会下调。携带编码无关基因如萤火虫荧光素酶或细菌β - 半乳糖苷酶的cDNA的结构相同的载体，其报告基因活性未表现出固醇调节。固醇对WHHL成纤维细胞以及感染了LDL受体逆转录病毒载体的WHHL成纤维细胞中的胰岛素受体活性也没有影响。用含有相同LDL受体cDNA的非逆转录病毒载体转染缺乏LDL受体的中国仓鼠卵巢（CHO）细胞，也导致转导的LDL受体对固醇产生反应。这些实验表明，固醇的作用对LDL受体转录本具有特异性。受感染细胞中转基因LDL受体mRNA水平不受固醇影响，这表明固醇介导的LDL受体活性降低并非由稳态mRNA水平的改变所致。这些数据表明存在负责转导的LDL受体基因表达的固醇调节的转录后水平机制。
