Mitochondrial localization of estrogen receptor beta

Abstract

Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ERalpha and ERbeta. ERalpha plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ERbeta is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ERbeta localizes to the mitochondria. In immunocytochemistry studies, ERbeta was detected with two ERbeta antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ERbeta and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ERbeta into the nucleus on 17beta-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ERbeta, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS/PAGE. The 50,000-65,000 M(r) band was digested with trypsin and subjected to matrix-assisted laser desorption/ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ERbeta. In summary, this study demonstrated that ERbeta is localized to mitochondria, suggesting a role for mitochondrial ERbeta in estrogen effects on this important organelle.

Fig. 1.

Fluorescence microscopy localization of ERβ to mitochondria in primary rat hippocampal neurons. (A) ERβ stained by using ERβ antibody, H150 (green). (B) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (C) Merged image of ERβ and mitochondria (yellow).

Fig. 2.

Fluorescence microscopy localization of ERβ to mitochondria in rat primary cardiomyocytes. (Aa) ERβ stained by using ERβ antibody, H150 (green). (Ab) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (Ac) Merged image of ERβ and mitochondria (yellow). (Ba)ERβ stained by using ERβ antibody, Z8P (green). (Bb) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (Bc) Merged image of ERβ and mitochondria (yellow).

Fig. 3.

Confocal microscopy of the colocalization of ERβ (H-150) with MitoTracker Red in HT-22 cells. ERβ was stained with ERβ (H150) as green, mitochondria with MitoTracker Red as red, and the merged image of ERβ and mitochondria as yellow. Cells were treated with DMSO (Left)or17β-estradiol (17-beta-E2, 10 nM) (Right) for 0.5 h. No nuclear translocation of ERβ was indicated after 17β-estradiol treatment. No ERβ staining was seen when H150 was omitted (data not shown).

Fig. 4.

Identification of ERβ in mitochondrial preparation. (A) MnSOD and histone H1 were used as a mitochondrial and a nuclear marker, respectively. Strong signals for ERβ and MnSOD were evident, but no nuclear (histone H1) signal was seen in the mitochondrial lysate (lane a). In cerebral lysate (lane b), ERβ, MnSOD, and histone H1 were detected. (B) Full-length hrERβ was used as positive control (lane b). A band with the same molecular weight as hrERβ was seen in both human heart homogenate (lane c) and purified human heart mitochondria (lane d). Biotinylated protein standards (lane a) were used as molecular weight markers. M_r values of 80,000, 60,000, and 50,000 are shown.

Fig. 5.

MALDI-TOF mass spectrum obtained from the M_r of 50,000–65,000 SDS/PAGE band of the isolated human heart mitochondria after in-gel digestion with trypsin. The major proteins identified by the database search are indicated in the spectrum, and the tryptic peptides that matched to those derived from sequence information of the ER proteins in the National Center for Biotechnology Information database are presented in Tables 1 and 2.