Mitochondrial localization of estrogen receptor ␤ Shao-Hua Yang*, Ran Liu*, Evelyn J. Perez*, Yi Wen*, Stanley M. Stevens, Jr.†, Thomas Valencia‡, Anne-Marie Brun-Zinkernagel§, Laszlo Prokai†, Yvonne Will¶, James Dykens*, Peter Koulen*, and James W. Simpkins*储 Departments of *Pharmacology and Neuroscience, ‡Integrative Physiology, and §Anatomy and Cell Biology, University of North Texas Health Science Center, Fort Worth, TX 76107; †Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32601; and ¶Pfizer La Jolla, 10624 Science Center Drive, San Diego, CA 92121 Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved January 26, 2004 (received for review October 24, 2003)

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: ER␣ and ER␤. ER␣ plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ER␤ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ER␤ localizes to the mitochondria. In immunocytochemistry studies, ER␤ was detected with two ER␤ 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 ER␤ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ER␤ into the nucleus on 17␤-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ER␤, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS兾PAGE. The 50,000 – 65,000 Mr band was digested with trypsin and subjected to matrix-assisted laser desorption兾ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ER␤. In summary, this study demonstrated that ER␤ is localized to mitochondria, suggesting a role for mitochondrial ER␤ in estrogen effects on this important organelle. nuclear receptor 兩 mitochondria

E

strogens play an important role in development, growth, and differentiation of both female and male secondary sex characteristics. Estrogen receptors (ERs) were the first identified nuclear receptor family member (1). The first ER, now called ER␣, was cloned in 1986 (2, 3). A second ER␤, was identified and cloned a decade later (4, 5). Like other members of the nuclear receptor superfamily, both ERs have a modular structure consisting of distinct functional domains (1). The DNA-binding domain (DBD) enables the receptor to bind its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AGGTCA spaced by 3 bp, referred to as an estrogen response element (ERE). The ligand-binding domain enables estrogen binding to the receptors. ERs are highly conserved between ER␣ and ER␤, with ⬎95% homology for the DBD and ⬇50% homology for the ligand-binding domain. Less homology is observed for the transactivational domain between ER␣ and ER␤ (5, 6). Genomic actions of ER␣ are well described (7). On binding to ER␣, estrogens induce a conformational change in the ER␣ proteins, which is accompanied by the dissociation of the accessory protein, heat shock protein 90, thereby exposing the DBD. In the nucleus, the receptor–ligand complex binds to DNA and modulates gene transcription. This transcriptional兾translational activation is comparatively slow and sensitive to cycloheximide and actinomycin D (8). The high homology of ER␤ to ER␣ in their DBD and ligand-binding domain indicates that these receptors may regulate common gene networks and respond to similar ligands. It is clear that ER␣ plays a major role as a transcription factor in the reproductive tissues for both male and female, and both male and female ER␣ knockout mice are infertile (9, 10). On the other hand, 4130 – 4135 兩 PNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12

little change of the transcription pattern in the reproductive tissue in the ER␤ knockout mice is seen (11). Increasing evidence supports nonclassical modes of ER action. Nongenomic actions of estrogens include cellular calcium homeostasis, induction of nitric oxide synthesis, and rapid activation of extracellular signal-regulated protein兾mitogen-activated protein kinase pathways (8, 12). A membrane ER that colocalizes with caveolin 1 has been described, which is identical with ER␣ and involved in the activation of endothelium nitric oxide synthase and extracellular signal-regulated protein (13). Also, a putative plasma membrane-associated ER-X has been proposed, which is associated with estradiol-induced activation of the mitogen-activated protein kinase cascade (14). Similarly, a subpopulation of ER␤ has also been localized in the plasma membrane of endothelial cell, which is involved in endothelium nitric oxide synthase signaling (15). Other cytoplasmic organelles have been described as containing ERs. Specific binding of estrogens to sites in the mitochondria has been described (16). Estrogens have consistently been indicated to modulate mitochondrial function, such as ATP production, mitochondrial membrane potential, and calcium concentration, although it is still unclear whether these actions are ER-dependent (17, 18). In transfected cells, ER␤ localizes to the nucleus (19), whereas the nuclear localization of ER␤ in nontransfected cells is rarely reported. Further, ER␤ is a poor transcriptional factor (20–24). The nuclear targeting of ER␣ is regulated cooperatively by multiple signals located in its hinge region (25). However, the hinge domain is one of the least conserved regions in ER␤ compared with ER␣ (4, 5). The unique structural characteristics of the hinge domain of ER␤ may lead to the differential intracellular targets of the receptor. In this study, we used immunocytochemistry, immunoblotting, and MS to demonstrate that ER␤ is localized to mitochondria. Our data establish this ER␤ localization in a variety of cell types, suggesting that estrogens can directly affect mitochondrial function through ER␤. Materials and Methods Chemicals and Reagents. 17␤-Estradiol was obtained from Ster-

aloids (Wilton, NH). Tissue culture material, Laemmli sample buffer, human recombinant ER␤ (hrER␤, long form), DTT, Coomassie brilliant blue, 20% Tris-glycine gel, and SeeBlue Plus 2 protein standard mixture were obtained from Invitrogen. Charcoalstripped FBS was from HyClone. MitoTracker Red, SlowFade Light Antifade reagent, and Alexa Fluor 488 goat anti-rabbit IgG were from Molecular Probes. ER␤ (H-150) (Santa Cruz Biotechnology) is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–150 mapping at the N This paper was submitted directly (Track II) to the PNAS office. Abbreviations: ER, estrogen receptor; DBD, DNA-binding domain; hrER␤, human recombinant ER␤; ERE, estrogen response element; ERR, ER-related protein; DAPI, 4⬘,6-diamidino2-phenylindole; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MnSOD, manganese superoxide dismutase. 储To

whom correspondence should be addressed at: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail: [email protected].

© 2004 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0306948101

Cell Culture. Primary cerebral cortical and hippocampal neurons.

Sprague–Dawley rat embryos (18 days old; from Charles River Breeding Laboratories) were externalized under halothane anesthesia. The cerebral cortex and hippocampus were dissected and harvested in 2 ml of preparation medium (DMEM, 4.5 g/liter glucose兾100 units/ml penicillin兾100 ␮g/ml streptomycin). The cortex and hippocampus were treated with trypsin. The tissue was washed three times with washing medium (Hanks’ medium, 4.5 g/liter glucose兾100 units/ml penicillin兾100 ␮g/ml streptomycin), and individual cells were isolated by trituration 10 times with three different sizes of fire-polished Pasteur pipettes. The cells were harvested in seeding medium (DMEM, 4.5 g/liter glucose兾100 units/ml penicillin兾100 ␮g/ml streptomycin兾2 mM glutamine兾19% horse serum) and filtered through a 40-␮m filter. The cerebral cortical cells and hippocampal cells were seeded in eight-well poly-L-lysine-treated chamber slide at the density of 20,000 cells per well. The cells were incubated in neurobasal medium (DMEM, 4.5 g/liter glucose兾100 units/ml penicillin兾100 ␮g/ml streptomycin兾2 mM glutamine, B27) in normal cell culture conditions. The cells were subjected to immunocytochemistry staining at day 7. Primary cardiomyocyte. Primary cardiomyocyte cultures were prepared from 2- to 4-day-old Sprague–Dawley rats. Cultures were maintained in medium 199 supplemented with 10% FBS (26). Murine hippocampus cell line. HT-22 cells (gift from D. Schubert, Salk Institute, San Diego), which are an immortalized murine hippocampal cell line, were maintained in DMEM media supplemented with 10% charcoal-stripped FBS and 20 ␮g兾ml gentamycin at 37°C in a humid atmosphere with 5% CO2. HT-22 cells (passages 18–25) were seeded into eight-well chamber slides at a density of 9,000 cells per well. Immunofluorescence Staining and Microscopy. Monolayer cells were

washed with PBS (pH 7.4) and fixed with cold methanol for 15 min at ⫺20°C. Cells were rinsed several times in PBS and incubated in ice-cold 0.2% Triton X-100 for 10 min to permeabilize the cells. Nonspecific sites were blocked for 1 h at room temperature with 5% normal goat serum and 5% BSA in PBS. Cells were then incubated with an ER␤ antibody (H-150 or Z8P) at 1:50 dilution at 4°C overnight. The sections were washed for 30 min in PBS, then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:200) in 5% normal goat serum with 5% BSA in PBS for 1 h at room temperature. After washing in PBS for 30 min the cells were stained with 100 ␮M 4⬘,6-diamidino-2-phenylindole (DAPI) for 5 min. Cells were mounted with SlowFade Light Antifade reagent and covered with a coverslip. Three controls were included in each experiment, in which we omitted the primary or secondary antibody or MitoYang et al.

Tracker Red. Samples were analyzed with either an Olympus microscope with appropriate excitation兾emission filter pairs or a Zeiss LSM confocal microscope. Purification of Mitochondria from Human Heart and Immunoblots of ER␤. Human heart mitochondria were isolated by differential

centrifugation from three donor hearts (obtained from Analytical Biological Services, Wilmington, DE). The donors were between 16 and 64 years of age and showed no indication of cardiovascular disease. Mitochondria (40 mg total) were further purified by metrizamide gradient centrifugation (27), and their integrity and purity were assessed by Western blot analysis for several proteins, including actin, dynamin, prohibitin, and the unique endoplasmic reticulum-resident C-terminal sequence (KDEL), which is required for the retention of proteins in the endoplasmic reticulum (28). Mitochondrial preparations were not made from any other cell types in this study. The extracted human heart mitochondria were combined in Laemmli buffer with ␤-mercaptoethanol and boiled for 5 min. Of the mitochondrial samples 30 ␮g were separated by 10% Trisglycine polycrylamide gel (Gradipore, Frenchs Forest, Australia) and then transferred to a nitrocellulose membrane (Millipore). Lanes containing biotinylated protein standards (Cell Signaling Technology, Beverly, MA) or Kaleidoscope prestained standards (Bio-Rad) were used to evaluate the size of the bands detected with ER␤. As a positive control, rat cerebral lysate or hrER␤ (long form) was assessed. The membranes were blocked for 1 h with 5% nonfat dry milk in PBS and were incubated overnight at 4°C with ER␤ antibody (H-150) at a dilution of 1:1,000. The membranes were repeatedly washed with PBS before incubation with a secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Bio-Rad) at 1:5,000 in PBS. The blots were developed with an enhanced chemiluminescent kit (Pierce). Identification of ER␤ in Mitochondria by MS. For SDS兾PAGE separation, the mitochondrial fraction was combined with 2⫻ Laemmli sample buffer plus DTT (0.125 M Tris䡠HCl兾4% SDS兾40% glycerol兾0.1% bromophenol blue, pH 6.8兾9 mM DTT) and heated at 90°C for 10 min. Mitochondrial protein (60 ␮g) was then loaded into each well of a 4–20% gradient Tris-glycine gel for protein separation. For molecular weight calibration, 10 ␮l of SeeBlue Plus 2 protein standard mixture was added to the first well. After protein separation, the gel was stained overnight with 0.1% Coomassie brilliant blue R-250 (45% methanol兾10% acetic acid). The gel was then placed in 5% acetic acid兾20% methanol (vol兾vol) to remove excess Coomassie brilliant blue that remained in the gel. A band that corresponded to molecular weights between 50,000 and 65,000 was excised from the gel, and the proteins were digested in-gel with trypsin as described by the Howard Hughes Medical Institute兾Keck Facility at Yale University (http:兾兾info.med.yale.edu兾wmkeck兾 prochem兾geldig3.htm). The digested mitochondrial proteins from the gel were purified with ZipTip microcolumns (Millipore) for matrix-assisted laser desorption ionization (MALDI)–time-offlight (TOF) MS. The tryptic peptides were concentrated onto a C18 ZipTip microcolumn, washed several times with 0.1% trifluoroacetic acid, and eluted off the column onto the MALDI plate with 1 ␮l of matrix solution. The matrix solution used was prepared by dissolving 10 mg of ␣-cyano-4-hydroxycinnamic acid in 1 ml of 60% acetonitrile兾0.1% trifluoroacetic acid. MALDI mass spectra were acquired on a Voyager DE-Pro MALDI-TOF mass spectrometer (Applied BioSystems) operated in reflector mode. Ions were accelerated by 20 kV after an extraction delay time of 200 ns. Grid and guide wire voltages were adjusted to 72% and 0.01% of the acceleration voltage value, respectively. MALDI-TOF兾MS values were a signal average of 50–100 laser shots and were mass-calibrated by using a four-point external calibration or internally calibrated (when applicable) to minor trypsin autolysis peaks at m兾z 842 and 2,211. Peptide mass values obtained by the MALDIPNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12 兩 4131

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terminus of ER␤ of human origin. It reacts with ER␤ of mouse, rat, and human origin by Western blotting, immunoprecipitation, and immunohistochemistry, and it is not cross-reactive with ER␣. Z8P (Zymed) is an epitope-affinity-purified rabbit antiserum raised against an 18-aa synthetic peptide (468–485, CSTEDSKSKEGSQNLQSQ) derived from the C terminus of mouse ER␤ protein. HPLC-grade acetonitrile, acetic acid, methanol, trifluoroacetic acid, and Optima water were purchased from Fisher Scientific. Sequencing-grade, modified trypsin along with the stabilityoptimized dilution buffer were obtained from Promega. Ultrapure SDS was purchased from Schwarz兾Mann. Ammonium bicarbonate and ␣-cyano-4-hydroxycinnamic acid were obtained from Sigma. For immunocytochemistry studies 17␤-estradiol was initially dissolved in DMSO and diluted in DMEM media to the final concentration of 10 nM with final DMSO concentration ⬍0.001%. For 17␤-estradiol treatment, cells were treated with 17␤-estradiol at 10 nM or 0.001% DMSO for 30 min, respectively. For mitochondrial labeling, cells were treated with 100 nM MitoTracker Red for 30 min in growth media. Then the media were removed, and immunostaining was done according to the manufacturers’ protocol.

Fig. 1. Fluorescence microscopy localization of ER␤ to mitochondria in primary rat hippocampal neurons. (A) ER␤ stained by using ER␤ antibody, H150 (green). (B) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (C) Merged image of ER␤ and mitochondria (yellow).

TOF兾MS analysis of in-gel tryptic digests were input into the MS-FIT search program of Protein Prospector (http:兾兾prospector.ucsf.edu). The National Center for Biotechnology Information (NCBI) nonredundant (nr) database was used in the appropriate program search options for protein identification. Results Mitochondrial Localization of ER␤ in Primary Neurons. To determine

the purity of the neuronal culture, primary cortical and hippocampal neurons were stained with mitogen-activated protein 2, glial fibrillary acidic protein, and DAPI. A specific neuronal marker (mitogen-activated protein 2 staining) was seen in cells with a neuronal morphology, whereas no evidence of glia (glial fibrillary acidic protein staining) was seen (data not shown). MitoTracker Red is a mitochondrion-selective dye that is well retained during cell fixation. Primary hippocampal neurons were costained with ER␤ (H150) and MitoTracker Red and were evaluated by using fluorescence microscopy. ER␤ staining was predominantly in the cytosol and presented a punctuate distribution, similar to that of mitochondria. In primary hippocampal neurons, ER␤ staining colocalized with MitoTracker Red. Other, yet unidentified cytosolic components were also stained with the antibodies used in these studies (Fig. 1). For primary cortical neurons, a similar colocalization of these two markers was seen (data not shown). To be certain that the two colors generated from the same mitochondria were not imaging artifacts caused by reciprocal detection of light emitted by MitoTracker Red or Alexa Fluor 488 (bleed-through), we omitted either MitoTracker Red or Alexa Fluor 488 conjugated secondary antibody, then performed immunocytochemistry. No signal was detected when cells were stained with MitoTracker Red and imaged for Alexa Fluor 488 and vice versa. Mitochondrial Localization of ER␤ in Primary Cardiomyocytes. To

determine whether this apparent mitochondrial localization of ER␤ was limited to neurons, colocalization of ER␤ and mitochondrial 4132 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0306948101

Fig. 2. Fluorescence microscopy localization of ER␤ to mitochondria in rat primary cardiomyocytes. (Aa) ER␤ stained by using ER␤ antibody, H150 (green). (Ab) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (Ac) Merged image of ER␤ and mitochondria (yellow). (Ba) ER␤ stained by using ER␤ antibody, Z8P (green). (Bb) Mitochondria stained with MitoTracker Red (red) and nuclei stained with DAPI (blue). (Bc) Merged image of ER␤ and mitochondria (yellow).

markers was assessed in primary cardiomyocytes. In primary cardiomyocytes, ER␤ (H-150) staining exhibited a punctuate cytoplasmic distribution, which colocalized with MitoTracker Red (Fig. 2A). A second antibody, Z8P, which detects 18 aa in the C terminus of ER␤, showed similar punctuate staining that also colocalized with MitoTracker Red (Fig. 2B). Mitochondrial Localization of ER␤ in HT-22 Cells by Confocal Microscopy. In HT-22 cells, ER␤ H-150 immunostaining exhibited a

granular cytoplasmic distribution regardless of the fixative used (acetone兾methanol or paraformaldehyde). The staining of the ER␤ coincided precisely with MitoTracker Red staining (Fig. 3). Moreover, no translocation of ER␤ into nucleus on 17␤-estradiol treatment for 0.5 h was seen (Fig. 3). Further, no ER␤ signal was seen in the cells in which the primary antibody was omitted (data not shown). Immunoblot of ER␤ in Purified Mitochondria from Human Heart. To confirm our immunocytochemistry study, immunoblots of ER␤ were performed with purified human heart mitochondrial protein. The nuclear marker (histone H1) was not observed, intense staining for the mitochondrial enzyme manganese superoxide dismutase (MnSOD) was evident, and a band with a molecular weight of 60,000 that reacted with the ER␤ antibody, H-150, was seen in the mitochondrial lysate (Fig. 4A). Rat cerebral lysate was used as positive control. MnSOD and histone H1 were seen in the cerebral lysate (Fig. 4A). As an additional positive control, we used fulllength hrER␤, and a band with the molecular weight of ⬇60,000 that stained with ER␤ H150 was seen for both human heart homogenate and purified human heart mitochondria (Fig. 4B). ER␤ was more concentrated in the mitochondrial preparation than Yang et al.

Fig. 4. Identification of ER␤ in mitochondrial preparation. (A) MnSOD and histone H1 were used as a mitochondrial and a nuclear marker, respectively. Strong signals for ER␤ and MnSOD were evident, but no nuclear (histone H1) signal was seen in the mitochondrial lysate (lane a). In cerebral lysate (lane b), ER␤, MnSOD, and histone H1 were detected. (B) Full-length hrER␤ was used as positive control (lane b). A band with the same molecular weight as hrER␤ 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. Mr values of 80,000, 60,000, and 50,000 are shown.

Fig. 3. Confocal microscopy of the colocalization of ER␤ (H-150) with MitoTracker Red in HT-22 cells. ER␤ was stained with ER␤ (H150) as green, mitochondria with MitoTracker Red as red, and the merged image of ER␤ and mitochondria as yellow. Cells were treated with DMSO (Left) or 17␤-estradiol (17-beta-E2, 10 nM) (Right) for 0.5 h. No nuclear translocation of ER␤ was indicated after 17␤-estradiol treatment. No ER␤ staining was seen when H150 was omitted (data not shown).

in the whole lysate. In a separate experiment, immunoblot of ER␤ was performed by using purified human heart mitochondrial protein from a different supplier (Molecular Probes), and the same results were obtained (data not shown). Identification of ER␤ in Human Heart Mitochondria by MS. Gel electrophoresis monitored by in-gel protease (trypsin) digestion and MALDI-TOF兾MS and sequence database searching were used for protein identification (29, 30). Identification of membrane or membrane-bound proteins such as receptors, transport channels, and ectoenzymes is difficult by using 2D gel electrophoresis. To circumvent this shortcoming, we used a 1D (SDS兾PAGE) separation before trypsin digestion and MS. A section of the gel containing protein with an apparent relative molecular weight (Mr) of 50,000– 65,000 and, therefore, expected to contain the ER␤, was excised, digested in-gel with trypsin, and analyzed by MALDI-TOF兾MS. As indicated in Fig. 5, several tryptic peptides were generated and were easily resolved for database search. Consistent with our immunoblots, numerous intense tryptic fragments of ATP synthase were found in our mitochondrial lysate, which indicated high purity of the mitochondrial preparation. In addition, monoisotopic masses input into MS-FIT also matched to several tryptic peptides derived from human ER␤ proteins, as shown in Tables 1 and 2. The sequences Yang et al.

Discussion Four major observations are reported in this study. First, ER␤ is localized to the mitochondria in several cell types. Second, this mitochondrial localization is independent of the differentiation state of cells, because it is seen in differentiated primary neurons and cardiac myocytes and in the undifferentiated HT22 cells and human lens epithelial cells (unpublished observations). Third, ER␤ does not translocate to the nucleus on exposure to its natural ligand, 17␤-estradiol. Finally, the mitochondrial localization can be demonstrated by using immunocytochemistry, immunoblotting, and MS. In MS, a large number of ER␤ specific epitopes were identified. Collectively, these results indicate that ER␤ is a component of the mitochondria and not primarily a nuclear transcriptional factor. The identification of ER␤ in mitochondria by using antibodies directed against both C-terminal and N-terminal amino acids in sequences that subsequently were identified by MS of purified mitochondria strongly support the conclusion that ER␤ is a mitochondrial protein. This observation may explain previous reports that ER␤ is a poor transcriptional factor. Studies with mice carrying disrupted ER genes indicated that ER␣ mediates the major proliferative effects of estrogen. In ER␣ knockout mice, a significant transcription pattern change is evident, such as hypotrophy of the uterus, ovary, testis, mammal gland, and vagina (9, 10). In contrast, both male and female ER␤ knockout mice show little change in their reproductive phenotype (11), suggesting that ER␤ is not a major transcriptional factor in reproductive tissues. Other evidence also suggests that ER␤ is a poor transcription factor. ERs regulate gene expression in two ways. ERs activate transcription after binding to ERE in the promoter region of estrogen-response genes. An alternative pathway has been reported in which ERs appear to be able to stimulate transcription from promoters that contain AP-1, Sp1, and cAMP response elements PNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12 兩 4133

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matched include both N-terminal and C-terminal epitopes to which the ER␤ antibodies, H150 and Z8P, respectively, are directed. H150 is directed to the fragment of N-terminal 150 amino acids, and we found fragments of 1–18 and 59–68. Z8P is directed to the fragment of C-terminal 18 aa (CSTEDSKSKEGSQNLQSQ), and SKEGSQNPQSQ was the tryptic peptide present. The DBD (147–154), ligand-binding domain (339–348), and hinge domain (179–191) sequences were also found in the matched sequences. The matched fragments were in fairly large amounts, indicating that these fragments are not likely the result of contamination from the nucleus. As shown in Table 2, eight sequences that matched with ER␤ isoform 3 were found in the mitochondrial lysate.

Fig. 5. MALDI-TOF mass spectrum obtained from the Mr of 50,000 – 65,000 SDS兾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.

sites. With regard to the ability to activate transcription with constructs that contain ERE, ER␤ is much weaker than ER␣ in most cell systems tested (20–24). Further, 17␤-estradiol binding to ER␣ but not ER␤ activates AP1 (31). Similarly, at Sp1 sites, ER␣ activates and ER␤ is nearly inactive, although both ERs physically interact with Sp1 (32). With an in vitro chromatin assembly and transcription system, ER␣ is a much more potent transcriptional activator than ER␤ (33). Induction of cyclin D1 gene transcription by ERs plays an important role in estrogen-mediated proliferation. Different action of ER␣ and ER␤ on cyclin D1 gene expression has been demonstrated. ER␣ activates cyclin D1 gene expression on binding to 17␤-estradiol, whereas ER␤ does not (34). We tested the possibility that the mitochondrial localization of ER␤ was the result of artifacts of our experimental procedure. The possibility that the mitochondrial preparations used were contaminated by other cellular organelles was addressed by using two stringent methods for the isolation of mitochondria and the further purification of mitochondrial proteins. This method yields protein fractions that are devoid of cytoskeleton marker proteins (actin and dynamin), the endoplasmic reticulum marker (KDEL), and the nuclear marker (histone), but contained abundant prohibitin, a mitochondrial marker. In this purified mitochondrial protein preparation, an Mr ⬇60,000 ER␤ antibody-interacting protein consistent with the long form of ER␤ was demonstrated by immunoblotting with two purified human heart preparations, one made by us and the other, a commercially available human heart mitochondrial protein (Molecular Probes). Further, seven fragments of ER␤ and eight fragments of ER␤-3 were identified by MS. Additionally, several identified fragments of ER␤ contain the epitopes to which the two ER␤ antibodies used are directed.

We also tested the possibility that the colocalization of ER␤ antibody binding with the mitochondrial marker MitoTracker Red was an artifact of bleeding through of emission light from one fluorophore into the detection range of the other. Staining of cells with only one fluorophore did not result in detection of light when the filter was set for the other fluorophore. Thus, the observed colocalization of ER␤ with a mitochondrial marker is not due to filter band-pass limitations and is seen in multiple neuronal and nonneuronal cell types. We assessed the possibility that the epitopes of ER␤ detected in mitochondria were from recently described ER-related proteins (ERRs). ERRs belong to orphan nuclear receptors. Three ERR (ERR1, ERR2, and ERR3) have been identified based on their sequence homology with the DBD of ERs (35, 36). The two ER␤ antibodies we used in this study are against N-terminal and Cterminal sequences, which share low homology with ER␣ and ERRs. We performed a BLAST search of GenBank with the epitopes of the two ER␤ antibodies. No hit was found in the ERR sequences. Therefore, it is unlikely that the antibodies we used in the immunocytochemistry and immunoblotting detected ERRs. A BLAST search of both N-terminal and C-terminal sequences identified by our MS consistently showed no match in the ERR sequences (data not shown). Mitochondria produce most of the cell’s ATP by oxidative phosphorylation and generate most of the endogenous oxygen radicals as a toxic by-product (37). In addition, mitochondria are central in the regulation of apoptosis, calcium homeostasis, and cytoplasmic redox state (38, 39). Estrogens have long been recognized as antioxidants, and recent studies have shown that estrogens are also potent neuroprotective agents (40–42). Several studies have also shown that estrogens may exert direct or indirect effects

Table 1. MS identification of ER␤ from purified mitochondrial protein m兾z submitted 993.5115 1165.5804 1189.6725 1215.6828 1260.6996 1442.7152 1922.0015

M ⫹ H⫹ matched 993.4722 1165.6329 1189.5449 1215.5428 1260.6775 1442.6810 1921.9078

⌬Mr 0.039 ⫺0.053 0.13 0.14 0.022 0.034 0.094

Peptide sequence (147–154) SCQACRLR, cysteine alkylation (59–68) SLEHTLPVNR (467–477) SKEGSQNPQSQ (181–191) SADEQLHCAGK, cysteine alkylation (339–348) LQHKEYLCVK (179–191) QRSADEQLHCAGK (1–18) MNYSIPSNVTNLEGGPGR, ox. methionine

Peptide fragments listed match the National Center for Biotechnology Information (NCBI) nonredundant (nr) database record numbers gi㛭7441774 and gi㛭1518263 for ER␤; Mr ⫽ 53,384. 4134 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0306948101

Yang et al.

Table 2. MS identification of ER␤3 from purified mitochondrial protein m兾z submitted 993.5115 1122.5952 1165.5804 1194.5684 1215.6828 1260.6996 1442.7152 1575.8009

M ⫹ H⫹ matched 993.4722 1122.5002 1165.6329 1194.5213 1215.5428 1260.6775 1442.6810 1575.7888

⌬Mr 0.039 0.095 ⫺0.053 0.047 0.14 0.022 0.034 0.012

Peptide sequence (147–154) SCQACRLR, cysteine alkylation (504–512) SFEACQQPR, cysteine alkylation (59–68) SLEHTLPVNR (467–477) SFEACQQPRE (181–191) SADEQLHCAGK, cysteine alkylation (339–348) LQHKEYLCVK (179–191) QRSADEQLHCAGK (477–489) LFMLREASCHGVR, cysteine alkylation

Peptide fragments listed match the National Center for Biotechnology Information (NCBI) nonredundant (nr) database record number gi㛭3091286 for ER␤3; Mr ⫽ 57,519.

on mitochondrial function. Estradiol can protect against ATP depletion, mitochondrial membrane potential decline, and the generation of reactive oxygen species induced by 3-nitroproprionic acid (17). 17␤-Estradiol can stabilize mitochondrial function against actions of mutant presenilin 1 (43) and modulate mitochondrial calcium influx (18). This study raises the possibility that mitochondrial ER␤ may mediate some or all of these actions of estrogen on mitochondrial function. Mitochondrial genes are potential sites of primary action of steroid hormones, and estrogens could directly modulate mitochondrial gene expression (44). The 16-kb mitochondrial genome encodes only 13 of the ⬎100 proteins involved in oxidative phosphorylation, and little is known about the regulation of mitochondrial gene expression (45). The ERE is characterized by a 15-nt motif that consists of two hexads in a palindromic configuration that is separated by 3 nt (1). The mitochondrial genome contains sequences similar to ERE, which are represented mostly as halfpalindromes (44), raising the possibility that ER␤ could modulate mitochondrial gene expression through binding to the similar ERE sequences in the mitochondrial genome.

In this study, evidence from immunocytochemistry, immunoblot, and proteometry suggests that ER␤ is a mitochondrial component. However, other yet unidentified cytosolic components were also stained with the antibodies used in differentiated primary neurons, but not in differentiated primary cardiac myocytes or undifferentiated hippocampal HT-22 cells. Inasmuch as this study was not designed to determine the effects of the state of differentiation of an individual cell type on the distribution of ER␤, subsequent studies are needed to address this issue. In summary, our studies indicate that, in a variety of cell types, ER␤ is a mitochondrial protein rather than a nuclear receptor. Localization of ER␤ to mitochondria suggests that ER␤ could mediate estrogen’s effects at this organelle, including its ability to modulate calcium influx, ATP production, apoptosis, and freeradical species generation.

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PNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12 兩 4135

CELL BIOLOGY

This work was supported in part by National Institute on Aging Grants AG10485 and AG22550, National Institute of Neurological Disorders and Stroke Grant NS44765, and U.S. Army Grant DAMD 17-19-9473.