HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells.
HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells.
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DOI：
10.1210/mend.13.4.0264
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发表时间：
1999-04
影响因子：
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通讯作者：
Cheng Cheng Zhang-Cheng;Sacha A. Krieg;D. J. Shapiro
中科院分区：
文献类型：
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作者：
Cheng Cheng Zhang-Cheng;Sacha A. Krieg;D. J. Shapiro
雌激素受体（ER）的毒性阻碍了脊椎动物细胞系的发展，稳定表达大量的重组野生型ER。为了分离稳定表达表位标记的ER的HeLa细胞的克隆系，我们使用编码单个双顺反子mRNA的构建体，其中FLAG表位标记的人ER α（fER）从5 '-翻译起始位点翻译并融合到新霉素抗性基因，所述新霉素抗性基因从内部核糖体进入位点翻译。一种稳定的HeLa-ER阳性细胞系（HeLa-ER 1）产生1，300，000个fER分子/细胞（比MCF-7细胞大约多20倍的ER）。HeLa fER在体内具有生物活性，这是通过在17 β-雌二醇或反式-羟基他莫昔芬存在下细胞的快速死亡以及细胞系激活转染的含雌激素应答元件（ERE）的报告基因的能力来判断的。FLAG标记的ER通过免疫亲和层析与抗FLAG单克隆抗体在一个步骤中纯化至接近同质。纯化的fER对17 β-雌二醇的分布常数（KD）为0.45 nM。纯化的HeLa fER和粗核提取物中的HeLa fER对ERE表现出相似的KD值（分别为0.8 nM和1 nM），这比我们对使用杆状病毒系统表达的纯化ER测定的10 nM的KD低约10倍。HMG-1强烈刺激粗品和纯化的HeLa fER与ERE的结合（KD为0.25 nM）。在转染的HeLa细胞中，HMG-1表现出17 β-雌二醇依赖性反式激活的剂量依赖性刺激。在高水平转染的HMG-1表达质粒中，ER的反式激活变得部分不依赖配体，并且反式羟基他莫昔芬的反式激活增加了25倍以上。这些数据描述了一种系统，其中ER在HeLa细胞中稳定表达并易于纯化，对ERE表现出极高的亲和力，并表明HMG-1的细胞内水平可能限制ER作用。
Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER alpha (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (approximately 20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17 beta-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (KD) for 17 beta-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar KD values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the KD of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (KD of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.