Binding of MCF-7 cell mitochondrial proteins and recombinant human estrogen receptors α and β to human mitochondrial DNA estrogen response elements

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 ERa 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. (C) 2004 Wiley-Liss, Inc.