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.

Our previous studies have shown that 17beta estradiol (E2) enhances the transcript levels of mitochondrial DNA (mtDNA)-encoded genes and mitochondrial respiratory chain (MRC) activity via estrogen receptors (ER). Others have reported the presence of putative estrogen responsive elements (ERE) in human mtDNA (mtEREs) and detection of ERs in mitochondria of rat uterine and ovary cells. Recently, we demonstrated the E2-enhanced mitochondrial localization of ERalpha and ERbeta, and E2-induced mtDNA transcript levels in MCF-7 cells. In this study, we applied electrophoresis mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to determine if mitochondrial extracts, recombinant human ERalpha (rhERalpha), and rhERbeta interact with mtEREs. Using EMSAs, we observed that ER-containing mitochondrial extracts bound to mtEREs and the binding was enhanced by E2, whereas the binding of mitochondrial proteins from ERbeta-deficient cells was almost undetectable. Both rhERalpha and rhERbeta bound to the mtEREs and their binding was altered by their respective antibodies. However, the ERalpha antibodies did not alter the binding of MCF-7 cell mitochondrial extracts to mtEREs whereas the binding MCF-7 and MDA-MB-231 cell mitochondrial extracts to mtEREs was reduced by ERbeta antibody. These results suggest that the mtERE-bound mitochondrial protein is ERbeta. Using SPR, we observed the binding of both ERs to mtEREs and that the binding was increased by E2. These results indicate that the mitochondrial ERs can interact with mtEREs and suggest that they may be directly involved in E2 induction of mtDNA transcription.

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