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. 2010 Sep;24(9):1737-47.
doi: 10.1210/me.2010-0125. Epub 2010 Jul 21.

Ligand-independent antiapoptotic function of estrogen receptor-beta in lung cancer cells

Guangfeng Zhang  1 , Naveena Yanamala, Kira L Lathrop, Lin Zhang, Judith Klein-Seetharaman, Harish Srinivas
Affiliations

Ligand-independent antiapoptotic function of estrogen receptor-beta in lung cancer cells

Guangfeng Zhang et al. Mol Endocrinol. 2010 Sep.

Abstract

Recent studies have demonstrated the presence of estrogen receptor (ER)beta in the mitochondria in various cell types and tissues, but the exact function of this localization remains unclear. In this study, we have examined the function of mitochondrial ERbeta in non-small-cell lung cancer (NSCLC) cells. Down-regulation of ERbeta by short hairpin RNA constructs sensitized NSCLC cells to various apoptosis-inducing agents such as cisplatin, taxol, and etoposide. The increased growth inhibition and induction of apoptosis in ERbeta-knockdown cells was observed irrespective of estrogen treatment, suggesting a ligand-independent role of ERbeta in regulating the intrinsic apoptotic pathway. Further, ERbeta from the mitochondrial fraction physically interacted with the proapoptotic protein Bad, in a ligand-independent manner. Glutathione-S-transferase pull-down assays and molecular modeling studies revealed that the DNA-binding domain and hinge region of ERbeta, and the BH3 domain of Bad were involved in these interactions. Further investigations revealed that ERbeta inhibited Bad function by disrupting Bad-Bcl-X(L) and Bad-Bcl-2 interactions. Reintroduction of ERbeta in the mitochondria of ERbeta knockdown cells reversed their sensitivity to cisplatin. Overall, our results demonstrate a ligand-independent role of ERbeta in regulating apoptosis, revealing a novel function for ERbeta in the mitochondria.

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Figures

Figure 1
Figure 1
ERβ is localized to mitochondria in NSCLC cells. A, Nuclear, mitochondrial, and cytosolic fractions were prepared from NSCLC cells and analyzed for ERβ by Western blotting. Cox IV and α-tubulin served as controls for mitochondrial and cytosolic fractions, respectively. B, Confocal microscopy of 201T cells stained with antibodies against ERβ (green) and Mitotracker Red (red). Overlap is shown in yellow.
Figure 2
Figure 2
ERβ ShRNA cells exhibit increased growth inhibition to chemotherapeutic agents. A–C, Calu-6 cells (stably expressing GFP or ERβ ShRNA constructs) and (D and E) 201T cells (transfected with GFP or ERβ SiRNA oligos) were grown in phenol red-free media under estrogen-free conditions and treated with various concentrations of cisplatin, taxol, or etoposide for 2–4 d. Cell growth was measured by Brd-U incorporation. Values are represented as mean (±sd) from five identical wells. Statistical analysis was performed by one-way ANOVA (P < 0.05). All the experiments were performed more than three times, and a representative experiment is shown.
Figure 3
Figure 3
ERβ ShRNA cells exhibit increased apoptosis. A, Calu-6 GFP and ERβ ShRNA cells grown under estrogen-free conditions were treated with cisplatin (100 μm) for 24 h. Cells were stained with Annexin V and propidium iodide and analyzed by flow cytometry. The percentage of Annexin V-positive cells in the two right quadrants is indicated. B, After the indicated treatments, cells were stained with Hoechst, and fragmented nuclei were visualized by confocal microscopy. The fragmented nuclei are denoted by arrows. C, Calu-6 cells were treated with cisplatin (100 μm) or taxol (20 nm) for 24 h, and the presence of cleaved caspase-3/9 and PARP was analyzed by Western blotting.
Figure 4
Figure 4
ERβ coimmunoprecipitates with Bad. A, Western blotting analysis of various Bcl-2 family members in Calu-6 GFP and ERβ ShRNA cells. B, A549 cells were transfected with Flag-ERβ along with HA-Bad or HA-PUMA constructs. Immunoprecipitations were performed with anti-HA antibodies followed by Western blotting with anti-Flag antibodies. C, Coimmunoprecipitation assays in 293T cells transfected with Flag-ERβ, HA-Bad, and HA-PUMA constructs. D, Endogenous Bad from mitochondrial fraction was immunoprecipitated with control IgG or anti-Bad antibodies and Western blotted with anti-ERβ antibodies. Cox IV and α-tubulin served as controls for mitochondrial and cytosolic fractions, respectively. IP, Immunoprecipitation; WT, wild type.
Figure 5
Figure 5
GST pull-down assays demonstrate ERβ and Bad interactions. A (top panel), Immobilized GST or GST-Bad was incubated with 35S-labeled ERβ, and bound proteins were separated by SDS-PAGE and visualized by autoradiography. Bottom panel, Coomassie-stained gel of recombinant GST and GST-Bad proteins. B, Schematic diagram of various ERβ deletion mutants. C, GST pull-down assays between GST-Bad and 35S-labeled ERβ mutants. GST-Bad-bound ERβ proteins are denoted by solid arrows in the upper panel. D, GST pull-down assays between Bad and [35S]ERβ in the presence or absence of 17β-estradiol (E2) (100 nm). WT, Wild type.
Figure 6
Figure 6
Molecular modeling studies of ERβ-Bad complex. A, Predicted three-dimensional structure of human Bad with the BH3 domain shown in yellow. B, Computer modeling of ERβ DBD-Bad complex with ERβ and Bad shown in green and cyan, respectively. C, Schematic representation of Bad deletion mutants. D (top panel), GST pull-down assays between Bad mutants and 35S-labeled ERβ. Bottom panel, Coomassie-stained gel of recombinant GST-Bad proteins. WT, Wild type.
Figure 7
Figure 7
ERβ inhibits Bad-Bcl-XL and Bad-Bcl-2 interactions. A, 35S-labeled Bcl-XL was incubated with immobilized GST or GST-Bad. Where indicated, these assays were performed in the presence of equal amounts of recombinant proteins GST (lane 3), GST-ERβ (a.a. 145-530) (lane 4), or GST-ERβ (1-144) (lane 5), and bound complexes were resolved on SDS-PAGE and visualized by autoradiography. B, Similar GST pull-down experiments were performed using GST-Bad and [35S]Bcl-2. C (left panel), 293T cells were transfected with Flag-ERβ, HA-Bad, and V5-Bcl-XL constructs. Lysates were immunoprecipitated with anti-HA antibodies and Western blotted with anti-V5 antibodies. The blots were stripped and reprobed with anti-Flag antibodies. C (right panel), Similar immunoprecipitations were performed in 293T cells transfected with Flag-ERβ (1-144), HA-Bad, and V5-Bcl-XL constructs. D (left panel), Endogenous Bad from cytosolic fractions of Calu-6 GFP ShRNA and ERβ ShRNA cells were immunoprecipitated with anti-Bad antibodies, and immune complexes were Western blotted with antibodies against ERβ or Bcl-XL. D (right panel), Western blots of ERβ, Bad, and Bcl-XL using cytosolic lysates. IP, Immunoprecipitation.
Figure 8
Figure 8
ERβ targeted to mitochondria promotes resistance to cisplatin-induced apoptosis. A, Schematic diagram of Flag-ERβ-TM construct. B, Western blotting of Calu-6 wild-type (WT) and ERβ ShRNA cells transfected with Flag-ERβ-TM construct. Lysates were probed with antibodies against ERβ, Flag, or β-actin. C, Confocal microscopy of ERβ ShRNA cells transfected with Flag-ERβ-TM. Cells were stained with antibodies against Flag (green) and with Mitotracker Red. Overlap is seen in yellow. D, Calu-6 wild-type (WT) and ERβ ShRNA cells transiently transfected with GFP-TM or Flag-ERβ-TM constructs were grown in phenol red-free media under estrogen-free conditions and treated with cisplatin (20 μm) for 3 d. Cell growth was measured by Brd-U incorporation. Values are represented as mean (±sd) from five identical wells. Statistical analysis was performed by one-way ANOVA (*, P < 0.05).
Figure 9
Figure 9
Schematic representation depicting the effects of ERβ knockdown. A, In the absence of ERβ, Bad interacts with Bcl-XL to free Bax to oligomerize and initiate apoptosis. B, In the presence of ERβ, Bad is sequestered and prevented from interaction with Bcl-XL, thus inhibiting apoptosis.
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References

    1. Ascenzi P, Bocedi A, Marino M 2006 Structure-function relationship of estrogen receptor α and β: impact on human health. Mol Aspects Med 27:299–402 - PubMed
    1. Matthews J, Gustafsson JA 2003 Estrogen signaling: a subtle balance between ER α and ER β. Mol Interv 3:281–292 - PubMed
    1. Levin ER 2005 Integration of the extranuclear and nuclear actions of estrogen. Mol Endocrinol 19:1951–1959 - PMC - PubMed
    1. Hall JM, McDonnell DP 1999 The estrogen receptor β-isoform (ERβ) of the human estrogen receptor modulates ERα transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578 - PubMed
    1. Cowley SM, Parker MG 1999 A comparison of transcriptional activation by ERα and ERβ. J Steroid Biochem Mol Biol 69:165–175 - PubMed

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