Chen Jin Q, Delannoy Michael, Cooke Carol, Yager James D
Division of Toxicological Sciences, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Am J Physiol Endocrinol Metab. 2004 Jun;286(6):E1011-22. doi: 10.1152/ajpendo.00508.2003. Epub 2004 Jan 21.
We observed previously that estrogen treatment increased the transcript levels of several mitochondrial DNA (mtDNA)-encoded genes for mitochondrial respiratory chain (MRC) proteins and MRC activity in rat hepatocytes and human Hep G2 cells. Others have reported detection of estrogen receptors (ER), ERalpha and ERbeta, in mitochondria of rabbit ovarian and uterine tissue. In this study, we have extended these observations. Using cellular fractionation and Western blot with ERalpha- and ERbeta-specific antibodies, we observed that ERalpha and ERbeta are present in mitochondria of human MCF7 cells and that the mitochondrial ERalpha and ERbeta account for 10 and 18%, respectively, of total cellular ERalpha and ERbeta in 17beta-estradiol (E(2))-treated MCF7 cells. We also found that E(2) significantly enhanced the amounts of mitochondrial ERalpha and ERbeta in a time- and concentration-dependent manner and that these effects are accompanied by a significant increase in the transcript levels of mtDNA-encoded genes, i.e., cytochrome c oxidase subunits I and II. Moreover, we demonstrated that these E(2)-mediated effects were inhibited by the pure ER antagonist, ICI-182780, indicating the involvement of ERs. Using immunohistochemistry with confocal microscopy and immunogold electron microscopy, we demonstrated that ERalpha and ERbeta are located within the MCF7 cell mitochondrial matrix. Computer analysis identified a putative internal mitochondrial targeting peptide signal within human ERbeta, suggesting an inherent potential for ERbeta to enter mitochondria. These findings confirm the observations of others and provide additional support for this novel localization of the ERs and for a potentially important role of the ER in the regulation of mtDNA transcription.
我们之前观察到，雌激素处理可增加大鼠肝细胞和人肝癌Hep G2细胞中几种线粒体DNA（mtDNA）编码的线粒体呼吸链（MRC）蛋白基因的转录水平以及MRC活性。其他人报道在兔卵巢和子宫组织的线粒体中检测到雌激素受体（ER）、ERα和ERβ。在本研究中，我们扩展了这些观察结果。通过细胞分级分离以及使用ERα和ERβ特异性抗体的蛋白质印迹法，我们观察到ERα和ERβ存在于人MCF7细胞的线粒体中，并且在17β-雌二醇（E₂）处理的MCF7细胞中，线粒体ERα和ERβ分别占细胞总ERα和ERβ的10%和18%。我们还发现，E₂以时间和浓度依赖性方式显著增加线粒体ERα和ERβ的量，并且这些效应伴随着mtDNA编码基因（即细胞色素c氧化酶亚基I和II）转录水平的显著增加。此外，我们证明这些E₂介导的效应被纯ER拮抗剂ICI-182780抑制，表明ER参与其中。使用共聚焦显微镜免疫组织化学和免疫金电子显微镜，我们证明ERα和ERβ位于MCF7细胞线粒体基质内。计算机分析在人ERβ中鉴定出一个假定的线粒体内靶向肽信号，表明ERβ具有进入线粒体的内在潜力。这些发现证实了其他人的观察结果，并为ER的这种新定位以及ER在mtDNA转录调控中的潜在重要作用提供了额外支持。
