Zhao Helen, Hart Laura L, Keller Ulrike, Holth Laurel T, Davie James R
Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Canada R3E 0V9.
J Cell Biochem. 2002;86(2):365-75. doi: 10.1002/jcb.10215.
Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.
用绿色荧光蛋白（GFP）标记激素受体增加了我们对激素受体配体依赖性亚细胞转运的了解。然而，标记的激素受体表达对相应野生型激素受体和内源性基因表达的影响尚未得到研究。在本研究中，我们构建了一个在四环素诱导系统控制下稳定表达GFP标记的人雌激素受体α（ER）的MCF-7细胞系，以确定GFP-ER表达对细胞增殖以及内源性ER和激素反应基因表达的影响。此外，利用诱导系统确定GFP-ER蛋白和mRNA的配体依赖性周转率。我们的结果表明，GFP-ER表达不影响细胞周期。与配体无关，GFP-ER显著降低内源性ER mRNA和蛋白水平，提示ER对其表达进行负性自调节。顺铂交联研究表明，GFP-ER在原位与核DNA相关联，提示GFP-ER在雌激素反应元件处部分取代了ER。此外，GFP-ER表达不影响雌二醇诱导的激素反应基因c-myc和pS2的瞬时表达。
