Fulvestrant (ICI 182,780)-dependent Interacting Proteins Mediate Immobilization and Degradation of Estrogen Receptor-α
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m510809200
ISSN1083-351X
AutoresXinghua Long, Kenneth P. Nephew,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-α (ERα) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERα interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERα-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERα-interacting proteins, cytokeratins 8 and 18 (CK8·CK18). We determined, using ERα-activating function-2 mutants, that helix 12 (H12) of ERα, but not its F domain, is essential for fulvestrant-induced ERα-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERα immobilization/degradation, transient transfection assays were performed using wild type ERα,ERα with a mutated H12, and ERα with a deleted F domain. Of those, only the ERα H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERα degradation in CK8·CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERα degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERα-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERα immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERα to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERα turnover. The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-α (ERα) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERα interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERα-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERα-interacting proteins, cytokeratins 8 and 18 (CK8·CK18). We determined, using ERα-activating function-2 mutants, that helix 12 (H12) of ERα, but not its F domain, is essential for fulvestrant-induced ERα-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERα immobilization/degradation, transient transfection assays were performed using wild type ERα,ERα with a mutated H12, and ERα with a deleted F domain. Of those, only the ERα H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERα degradation in CK8·CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERα degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERα-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERα immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERα to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERα turnover. Estrogen receptor-α (ERα), 2The abbreviations used are: ERα, estrogen receptor-α; CK, cytokeratin; E2, 17β-estradiol; GFP, green fluorescent protein; ICI, ICI 182,780; 4-OHT, 4-hydroxytamoxifen; siRNA, small interference RNA; SERD, selective estrogen receptor down-regulator; GAPDH, glyceraldehyde phosphate dehydrogenase; GST, glutathione S-transferase; AF2, activating function-2; wt, wild type; H12, helix 12. a member of the nuclear receptor family, is a ligand-dependent transcription factor that mediates physiological responses to its cognate ligand, 17β-estradiol (E2), in estrogen target tissues such as the breast, uterus, and bone (1Barkhem T. Nilsson S. Gustafsson J.A. Am. J. Pharmacogenomics. 2004; 4: 19-28Crossref PubMed Scopus (82) Google Scholar). Because ERα is a short-lived protein (half-life of 4-5 h), its cellular levels are strictly regulated (2Eckert R.L. Mullick A. Rorke E.A. Katzenellenbogen B.S. Endocrinology. 1984; 114: 629-637Crossref PubMed Scopus (111) Google Scholar). Although ERα turnover is a continuous process (2Eckert R.L. Mullick A. Rorke E.A. Katzenellenbogen B.S. Endocrinology. 1984; 114: 629-637Crossref PubMed Scopus (111) Google Scholar), dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway (3Alarid E.T. Bakopoulos N. Solodin N. Mol. Endocrinol. 1999; 13: 1522-1534Crossref PubMed Google Scholar, 4El Khissiin A. Leclercq G. FEBS Lett. 1999; 448: 160-166Crossref PubMed Scopus (128) Google Scholar, 5Nawaz Z. Lonard D.M. Dennis A.P. Smith C.L. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1858-1862Crossref PubMed Scopus (495) Google Scholar, 6Lonard D.M. Nawaz Z. Smith C.L. O'Malley B.W. Mol. Cell. 2000; 5: 939-948Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar), occur in response to changing cellular conditions (7Reid G. Denger S. Kos M. Gannon F. Cell. Mol. Life Sci. 2002; 59: 821-831Crossref PubMed Scopus (128) Google Scholar, 8Fan M. Bigsby R.M. Nephew K.P. Mol. Endocrinol. 2003; 17: 356-365Crossref PubMed Scopus (108) Google Scholar, 9Fan M. Nakshatri H. Nephew K.P. Mol. Endocrinol. 2004; 18: 2603-2615Crossref PubMed Scopus (75) Google Scholar). In addition, differing ligands have been demonstrated to exert differential effects on steady-state levels of ERα (10Wijayaratne A.L. McDonnell D.P. J. Biol. Chem. 2001; 276: 35684-35692Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 11Preisler-Mashek M.T. Solodin N. Stark B.L. Tyriver M.K. Alarid E.T. Am. J. Physiol. Endocrinol. Metab. 2002; 282: 891-898Crossref PubMed Scopus (82) Google Scholar). For example, E2 and the "pure" ERα antagonists (i.e. ICI 164,384, ICI 182,780, RU 58,668, and ZK-703) (12Dauvois S. Danielian P.S. White R. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4037-4041Crossref PubMed Scopus (434) Google Scholar, 13Dauvois S. White R. Parker M.G. J. Cell Sci. 1993; 106: 1377-1388Crossref PubMed Google Scholar) induce receptor turnover, whereas the "partial" agonist/antagonist 4-hydroxytamoxifen (4-OHT) stabilizes ERα (14Wijayaratne A.L. Nagel S.C. Paige L.A. Christensen D.J. Norris J.D. Fowlkes D.M. McDonnell D.P. Endocrinology. 1999; 140: 5828-5840Crossref PubMed Google Scholar, 15Fan M. Park A. Nephew K.P. Mol. Endocrinol. 2005; 19: 2901-2914Crossref PubMed Scopus (155) Google Scholar). E2-mediated ERα degradation is dependent on transcription, coactivator recruitment, and new protein synthesis, whereas ICI-induced degradation of ERα is independent of these processes (16Reid G. Hubner M.R. Metivier R. Brand H. Denger S. Manu D. Beaudouin J. Ellenberg J. Gannon F. Mol. Cell. 2003; 11: 695-707Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 17Nardulli A.M. Katzenellenbogen B.S. Endocrinology. 1986; 119: 2038-2046Crossref PubMed Scopus (76) Google Scholar, 18Seo H.S. Larsimont D. Querton G. El Khissiin A. Laios I. Legros N. Leclercq G. Int. J. Cancer. 1998; 78: 760-765Crossref PubMed Scopus (33) Google Scholar). Thus, although both E2 and pure antiestrogens induce ERα degradation, their mechanisms of action differ markedly. In addition to altering ERα stability and turnover, different ligands have been shown to have profoundly distinct effects on receptor mobility and cellular localization. For example, ERα was found localized exclusively in the nucleus after E2 and 4-OHT treatment, whereas ICI caused both nuclear and cytoplasmic receptor localization (13Dauvois S. White R. Parker M.G. J. Cell Sci. 1993; 106: 1377-1388Crossref PubMed Google Scholar, 19Htun H. Holth L.T. Walker D. Davie J.R. Hager G.L. Mol. Biol. Cell. 1999; 10: 471-486Crossref PubMed Scopus (220) Google Scholar). Stenoien et al. (20Stenoien D.L. Patel K. Mancini M.G. Dutertre M. Smith C.L. O'Malley B.W. Mancini M.A. Nat. Cell Biol. 2001; 3: 15-23Crossref PubMed Scopus (341) Google Scholar), using fluorescence recovery after photobleaching, demonstrated that E2, 4-OHT, and ICI treatment resulted in reduced nuclear mobility of ERα tagged with cyan fluorescent protein (20Stenoien D.L. Patel K. Mancini M.G. Dutertre M. Smith C.L. O'Malley B.W. Mancini M.A. Nat. Cell Biol. 2001; 3: 15-23Crossref PubMed Scopus (341) Google Scholar). In that study complete fluorescence recovery was not observed after ICI treatment due to immobilization of ERα to the nuclear matrix (20Stenoien D.L. Patel K. Mancini M.G. Dutertre M. Smith C.L. O'Malley B.W. Mancini M.A. Nat. Cell Biol. 2001; 3: 15-23Crossref PubMed Scopus (341) Google Scholar). Additional studies have further shown a rapid immobilization of the ERα-ICI complex within the nuclear matrix, with sequestration in a salt-insoluble, nuclear compartment (21Giamarchi C. Chailleux C. Callige M. Rochaix P. Trouche D. Richard-Foy H. Biochim. Biophys. Acta. 2002; 1578: 12-20Crossref PubMed Scopus (15) Google Scholar, 22Callige M. Kieffer I. Richard-Foy H. Mol. Cell. Biol. 2005; 25: 4349-4358Crossref PubMed Scopus (74) Google Scholar), although the precise nature of the receptor-nuclear matrix interaction remains unknown. Fulvestrant (faslodex, ICI 182,780) belongs to a new class of antihormonal therapy for advanced breast cancer called selective estrogen receptor down-regulators (SERDs) (23Howell A. Eur. J. Cancer. 2000; 36 (Suppl. 4): 87-88Abstract Full Text Full Text PDF PubMed Google Scholar, 24McDonnell D.P. Clin. Cancer Res. 2005; 11: 871-877PubMed Google Scholar). SERDs act as potent antagonists by inducing rapid receptor turnover and display no agonist activity in estrogen target tissues. SERDs differ markedly from the class of molecules called selective estrogen receptor modulators (SERMs), such as 4-OHT, that function as either agonists or antagonists, depending upon the target tissue (24McDonnell D.P. Clin. Cancer Res. 2005; 11: 871-877PubMed Google Scholar). The pure antagonistic property of fulvestrant is due to a steroidal structure containing a long bulky side chain (25Pike A.C. Brzozowski A.M. Walton J. Hubbard R.E. Thorsell A.G. Li Y.L. Gustafsson J.A. Carlquist M. Structure (Camb). 2001; 9: 145-153Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), which induces a distinct conformational change in the ligand binding domain of ERα (26Wu Y.L. Yang X. Ren Z. McDonnell D.P. Norris J.D. Willson T.M. Greene G.L. Mol. Cell. 2005; 18: 413-424Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), specifically in the position of helix 12 (H12), to prevent receptor dimerization and binding to DNA (27Fawell S.E. White R. Hoare S. Sydenham M. Page M. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6883-6887Crossref PubMed Scopus (346) Google Scholar). Because specific mutations in H12 can reverse the pure antiestrogenic properties of fulvestrant (28Mahfoudi A. Roulet E. Dauvois S. Parker M.G. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4206-4210Crossref PubMed Scopus (113) Google Scholar, 29Montano M.M. Ekena K. Krueger K.D. Keller A.L. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 230-242Crossref PubMed Scopus (85) Google Scholar), H12 may contribute to fulvestrant-induced ERα degradation. In this study the mechanism of fulvestrant-induced ERα degradation by the ubiquitin-proteasome pathway was investigated. We show that this SERD induces specific ERα cytokeratins CK8·CK18 interactions, the major intermediate filament proteins found in the nuclear matrix and cytoplasm of ERα-positive breast cancer cell lines (30Coutts A.S. Davie J.R. Dotzlaw H. Murphy L.C. J. Cell. Biochem. 1996; 63: 174-184Crossref PubMed Scopus (38) Google Scholar). We further demonstrate that H12 is essential for these cytokeratin interactions and, subsequently, receptor immobilization within the nuclear matrix. Furthermore, we show that fulvestrant-mediated receptor degradation and cytoplasmic localization correlate directly with CK8 and CK18 levels in breast cancer cells. Because proteasomes have been shown to be associated primarily with intermediate filaments (31Olink-Coux M. Arcangeletti C. Pinardi F. Minisini R. Huesca M. Chezzi C. Scherrer K. J. Cell Sci. 1994; 107: 353-366Crossref PubMed Google Scholar, 32Arcangeletti C. Sutterlin R. Aebi U. De Conto F. Missorini S. Chezzi C. Scherrer K. J. Struct. Biol. 1997; 119: 35-58Crossref PubMed Scopus (51) Google Scholar, 33Arcangeletti C. De Conto F. Sutterlin R. Pinardi F. Missorini S. Geraud G. Aebi U. Chezzi C. Scherrer K. Eur. J. Cell Biol. 2000; 79: 423-437Crossref PubMed Scopus (19) Google Scholar), we suggest that fulvestrant induces specific receptor-cytokeratin interactions in the nuclear matrix, bringing ERα into close proximity to proteasomes for subsequent degradation. Materials—The following antibodies and reagents were used in this study: anti-ERα (HC20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or monoclonal anti-human ERα (Chemicon International, Inc., Temecula, CA); monoclonal anti-human cytokeratin 8 (RCK102; BD Biosciences) and monoclonal anti-human cytokeratin 18 (RCK106; BD Biosciences); monoclonal anti-cytokeratin peptide 8 (Sigma); mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH) (Chemicon International); glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences); SuperSignal West Pico chemiluminescent substrate (Pierce); protease inhibitor mixture set III (Calbiochem-Novabiochem); Lipofectamine Plus reagent, Geneticin, and cell culture reagents (Invitrogen); FuGENE (Roche Applied Science); 4-OHT and MG132 (Sigma); ICI 182,780 (Tocris Cookson Ltd., Ellisville, MO); RNase-free DNase I and BL21 (DE3)pLysS competent cells (Promega, Madison, WI). Plasmid Construction—Wild type ERα pSG5-ERα(HEGO) was kindly provided by Dr. Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) and GFP-ERα (26Wu Y.L. Yang X. Ren Z. McDonnell D.P. Norris J.D. Willson T.M. Greene G.L. Mol. Cell. 2005; 18: 413-424Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) by Dr. Michael Mancini (Baylor College of Medicine, Houston, TX). The ERα helix 12 mutant pRST-7-hER3X (D538N/E542Q/D545N) was kindly provided by Donald McDonnell (Duke University, Durham, NC). pGEX-6P-1-AF2, pGEX-6P-1-AF2ΔF, pGEX-6P-1-AF2ΔFΔH12, and pGEX-6P-1-ERα3X-AF2 were constructed by inserting the PCR DNA fragment of interest into pGEX-6P-1 (BamHI and XhoI site). pcDNA3-ERαΔF, pcDNA3-ERα3XΔF, and pcDNA3-ERαΔFΔH12 were generated by inserting the specific PCR DNA fragment into pcDNA3MycHisA (BamHI and XhoI site). pcDNA3-CK8 was generated by inserting the CK8 PCR DNA fragment into pcDNA3MycHisA (BamHI and XhoI site). pcDNA3-CK18 was generated by inserting CK18 PCR DNA fragment into pcDNA3MycHisA (EcoRI and XhoI site). Cloning results were confirmed by subjecting all constructs to DNA sequencing. Cell Lines—The human cervical carcinoma HeLa cell line and the breast cancer cell lines MCF-7 and its daughter, C4-12 (ERα-negative, CK8- and 18-positive (34Oesterreich S. Zhang P. Guler R.L. Sun X. Curran E.M. Welshons W.V. Osborne C.K. Lee A.V. Cancer Res. 2001; 61: 5771-5777PubMed Google Scholar)), are routinely maintained in our laboratory, as described previously (9Fan M. Nakshatri H. Nephew K.P. Mol. Endocrinol. 2004; 18: 2603-2615Crossref PubMed Scopus (75) Google Scholar, 35Fan M. Long X. Bailey J.A. Reed C.A. Osborne E. Gize E.A. Kirk E.A. Bigsby R.M. Nephew K.P. Mol. Endocrinol. 2002; 16: 315-330Crossref PubMed Scopus (47) Google Scholar). MDA-MB-231 and T47D breast cancer cells were purchased from ATCC (Manassas, VA). MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium with 50 units/ml penicillin, 50 μg/ml streptomycin, 10 mm Hepes, 6 ng/ml insulin, and 10% fetal bovine serum. T47D cells were maintained in RPMI 1640 medium 2 mm l-glutamine, 1.0 mm sodium pyruvate, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 mm Hepes, 0.2 units/ml insulin, and 10% fetal bovine serum. Before experiments involving transient transfection and hormone treatment, cells were cultured in hormone-free medium (phenol red-free minimum Eagle's medium (MEM) with 5% charcoal-stripped fetal bovine serum) for 3 days. Stable Transfection of ERα—C4-12 or HeLa cells were transfected with pcDNA-ERα (C4-12/ERα and HeLa/ERα, respectively) using Lipofectamine Plus Reagent and exposed to antibiotic (G418; 0.5 mg/ml) for 3 weeks. Expression of ERα in G418-resistant colonies was verified by immunoblotting with anti-ERα. Transient Transfection Assay—T47D and HeLa cells were cultured in hormone-free medium for 3 days and transfected with equal amounts of total plasmid DNA (adjusted by the corresponding empty vectors) using Lipofectamine Plus reagent or FuGENE according to the manufacturer's guidelines. Five hours later, the DNA/Lipofectamine mixture was removed, and cells were cultured in hormone-free medium. Unless stated otherwise, 24 h after transfection, cells were treated with the specified drug. RNA Interference (siRNA)—siRNA transfection reagent, control siRNA, CK8 siRNA, and CK18 siRNA were purchased from Santa Cruz Biotechnology. The CK8 and CK18 siRNAs (singly or both) were transfected into MCF-7 cells according to the manufacturer's protocol; 72 h after transfections, cells were treated with 100 nm ICI 182,780. Whole cell lysates were prepared in 1× SDS sample buffer. Protein levels were examined by Western blotting using specific antibodies. Preparation of Whole Cell Extracts—Whole cell extracts were prepared by suspending cells in SDS lysis buffer (62 mm Tris, pH 6.8, 2% SDS, 10% glycerol, and protease inhibitor mixture III). After 15 min of incubation on ice, extracts were sonicated, insoluble materials were removed by centrifugation (15 min at 12,000 × g), and supernatant protein concentrations were determined using a Bio-Rad protein assay kit. Preparation of Nuclear Extracts and Nuclear Matrix—Nuclear extract was prepared using a nuclear extraction kit (Active Motif, Carlsbad, CA), according to the manufacturer's protocol. Nuclear matrix was prepared following the procedure described by Coutts et al. (30Coutts A.S. Davie J.R. Dotzlaw H. Murphy L.C. J. Cell. Biochem. 1996; 63: 174-184Crossref PubMed Scopus (38) Google Scholar). Briefly, cell nuclei were extracted with nuclear matrix buffer (100 mm NaCl, 300 mm sucrose, 10 mm Tris-HCl, pH 7.4, 2 mm MgCl2, 1% (v/v) thiodiglycol) containing 1 mm phenylmethylsulfonyl fluoride and 0.5% (v/v) Triton X-100. Nuclei were resuspended in digestion buffer (50 mm NaCl, 300 mm sucrose, 10 mm Tris-HCl, pH 7.4, 3 mm MgCl2, 1% (v/v) thiodiglycol, 0.5% (v/v) Triton X-100), digested with DNase I (168 units/ml) for 20 min at room temperature, and then sequentially extracted using 0.25 m ammonium sulfate and 2 m NaCl. Nuclear matrix was resuspended in 1× SDS sample buffer and sonicated. Western Blot and Quantitation—Whole cell lysates were prepared in 1× SDS sample buffer by sonication, and total protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. ERα levels were determined by Western blot using a LI-COR (Lincoln, NE) imaging system. The membrane was incubated with primary antibody followed by incubation with infrared dye IR800-labeled goat anti-mouse IgG or IR700-labeled goat anti-rabbit IgG (LI-COR) secondary antibodies and quantitated with LI-COR Odyssey software. For immunoblotting by enhanced chemiluminescence (ECL), primary antibody was detected by horseradish peroxidase-conjugated second antibody and visualized using an enhanced SuperSignal West Pico chemiluminescent substrate. GST Pull-down Assay—GST pull-down assays were performed as we have described previously (35Fan M. Long X. Bailey J.A. Reed C.A. Osborne E. Gize E.A. Kirk E.A. Bigsby R.M. Nephew K.P. Mol. Endocrinol. 2002; 16: 315-330Crossref PubMed Scopus (47) Google Scholar, 36Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar). To fuse ERα-AF2 with GST, an ERαAF2 PCR fragment (amino acids 297-595) was cloned into the BamHI and XhoI sites of the plasmid pGEX-6P-1 and subjected to DNA sequencing to confirm the correct reading frame. The GST-tagged AF2 was then expressed in BL21 cells and purified as described (36Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar, 37Cavailles V. Dauvois S. Danielian P.S. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10009-10013Crossref PubMed Scopus (341) Google Scholar). Briefly, overnight cultures of BL21 cells containing the plasmid pGEX-6P-1-GST-ERα-AF2 were diluted (1:20), cultured in fresh medium for 2 h, and treated with 0.1 mm isopropyl β-d-thiogalactoside for 3 h. Induced bacteria were then collected by centrifugation and lysed in NETN buffer containing 0.5% Nonidet P-40, 1 mm EDTA, 20 mm Tris, pH 8.0, 100 mm NaCl, and protease inhibitors. GST-ERα-AF2 was purified on glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences). MCF-7 cell lysates were prepared by sonicating cells in cell lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, pH 7.5). Whole cell lysates were then incubated with the glutathione-bound GST-ERα-AF2 in binding buffer (60 mm NaCl, 1 mm EDTA, 20 mm Tris, pH 7.5, 0.05% Nonidet P-40, 1 mm dithiothreitol, 6 mm MgCl2, and 8% glycerol) in the absence or presence of corresponding ligands or vehicle for 3 h at 4 °C. After washing with binding buffer, ERα-AF2-bound proteins were eluted, separated by 10% SDS-polyacrylamide, and visualized by Coomassie Blue. Specific proteins were cut from the gel, eluted, and analyzed by MALDI and liquid chromatography mass spectrometry by the Indiana University Protein Analysis Research Center (Indianapolis, IN). Co-immunoprecipitation—MCF-7 cell whole cell lysates were prepared in lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% Triton X-100, 1 mm Na3VO4, protease inhibitor). Whole cell extract was incubated with protein G-agarose for 30 min at 4 °C. After centrifugation at 12,000 × g for 15 s, the precleared supernatants were incubated with 5 μl of anti-ERα antibody or IgG at 4 °C for 3 h followed by incubation with 30 μl of protein G-agarose beads for 30 min. The beads were then pelleted by brief centrifugation, washed 3 times with Tris-buffered saline (TBS) and once with TBS containing 0.4 m NaCl, and resuspended in 30 μl of SDS-PAGE loading buffer for SDS-PAGE and Western blotting. Live Cell Microscopy and Drug Treatment—Live fluorescence microscopy was performed by growing cells on 6-well plates and transfection with GFP-ERα using Lipofectamine or FuGENE and maintained in minimum Eagle's medium with 5% dextran-coated charcoal-stripped fetal bovine serum at 37 °C. Cells were treated with E2 (10 nm), ICI (100 nm), 4-OHT (100 nm), or ICI and cycloheximide (25 μg/ml). Images were taken using a Zeiss Axiovert 40 Inverted Microscope and Axio-Vision software. Fulvestrant Induces ERα-Intermediate Filament Protein Interactions—Previously it was shown that treatment of breast cancer cells with the pure antagonist ICI resulted in ERα immobilization and resistance to biochemical extraction within the nuclear matrix (21Giamarchi C. Chailleux C. Callige M. Rochaix P. Trouche D. Richard-Foy H. Biochim. Biophys. Acta. 2002; 1578: 12-20Crossref PubMed Scopus (15) Google Scholar). For this study we hypothesized that fulvestrant-dependent ERα-interacting proteins in the nuclear matrix were responsible for this phenomena. To identify putative fulvestrant-dependent ERα interacting proteins, cell lysates from human breast cancer MCF-7 cells were incubated with immobilized GST-ERα-activating function-2 (AF2) in the presence of ICI. Interacting proteins were eluted from the beads, separated by SDS-PAGE, and stained with Coomassie Blue. Fulvestrant-specific interacting protein bands (Fig. 1A) were excised from the gel and subjected to mass spectrometry (MALDI and liquid chromatography mass spectrometry) analysis, resulting in two of the proteins being identified as cytokeratins 8 and 18 (CK8 and CK18). To validate those findings, Western blot analysis using CK8- or CK18-specific antibodies, was performed to permit conclusive identification of these putative ERα binding partners (Fig. 1, B and C). No interaction between ERα and CK8 or CK18 was observed in the presence of either E2 or 4-OHT (Fig. 1). These ERα-CK8·CK18 associations were also stable in the presence of high salt (Fig. 1, last lane), consistent with other reports that ERα is insoluble after immobilization by ICI or RU 58668 (21Giamarchi C. Chailleux C. Callige M. Rochaix P. Trouche D. Richard-Foy H. Biochim. Biophys. Acta. 2002; 1578: 12-20Crossref PubMed Scopus (15) Google Scholar, 38Marsaud V. Gougelet A. Maillard S. Renoir J.M. Mol. Endocrinol. 2003; 17: 2013-2027Crossref PubMed Scopus (127) Google Scholar). To further demonstrate an ERα-CK8·CK18 interaction in vivo, co-immunoprecipitation was performed using MCF-7 whole cell lysates and an ERα-specific antibody in the absence or presence of fulvestrant. As shown in Fig. 1D, CK8 and CK18 were seen in the ERα complex only in the presence of ICI, suggesting that fulvestrant induces an endogenous interaction between ERα and CK8·CK18. Expression of CK8·CK18 in ERα-positive and -negative Cancer Cell Lines—It has been previously shown that both CK8 and CK18 are nuclear matrix-intermediate filament proteins present in ERα-positive cells (30Coutts A.S. Davie J.R. Dotzlaw H. Murphy L.C. J. Cell. Biochem. 1996; 63: 174-184Crossref PubMed Scopus (38) Google Scholar). To investigate whether a correlation exists between expression of ERα and/or CK8·CK18, whole cell lysates were prepared from human breast (MCF-7, T47D, MDA-MB-231) and cervical cancer (HeLa) cell lines. Levels of CK8·CK18 and ERα were determined by Western blot analysis. Differential CK expression was observed between the ERα-positive and -negative cell lines (Fig. 2A). Furthermore, CK8 and CK18 protein levels were markedly higher in MCF-7 and T47D (ERα-positive) cells as compared with the ERα-negative MDA-MB-231 and HeLa cells. Effect of Fulvestrant on the Association of ERα with the Nuclear Matrix and Receptor Degradation—Distinct ligands can specifically affect ERα extractability from the nucleus of breast cancer cells (38Marsaud V. Gougelet A. Maillard S. Renoir J.M. Mol. Endocrinol. 2003; 17: 2013-2027Crossref PubMed Scopus (127) Google Scholar). To further characterize the association between ERα and the nuclear matrix in the presence of antiestrogens, MCF-7 and T47D cells (ERα-, CK8-, and CK18-positive) were treated with ICI or 4-OHT followed by isolation of nuclear matrix fractions. Nuclear matrix prepared from MDA-MB-231 (ERα-negative; CK8- and CK18-positive, Fig. 2A) was used as a control. In the nuclear matrix of ERα-positive cells, CK8 and CK18 were highly abundant (Fig. 2C, upper panel, Coomassie Blue; middle panel, Western blot). In the presence of ICI, the majority of ERα protein was unextractable and remained tightly associated with the nuclear matrix (Fig. 2C); in contrast, in the presence of 4-OHT, ERα was loosely associated with the nuclear matrix, readily extractable, and thus, more abundant in the nuclear extract (Fig. 2C, bottom panel). These observations are consistent with the result that fulvestrant induces a salt-resistant ERα-CK8 and -CK18 interaction (Fig. 1) and that ERα extractability varies in the presence of different ligands (38Marsaud V. Gougelet A. Maillard S. Renoir J.M. Mol. Endocrinol. 2003; 17: 2013-2027Crossref PubMed Scopus (127) Google Scholar). To monitor ERα immobilization and degradation, nuclear extract and nuclear matrix were prepared from MCF-7 cells treated with fulvestrant for 0-4 h. As shown in Fig. 2B, rapid (<30 min) immobilization of ERα from the nuclear extract to the nuclear matrix was observed followed by receptor degradation 1 h after the onset of ICI treatment. In addition, CK8 and CK18 were both localized in the insoluble nuclear matrix (Fig. 2B). Taken together, these observations demonstrate that after treatment with fulvestrant, ERα is rapidly sequestered in a salt-insoluble nuclear compartment before being degraded. Helix 12 Is Required for Fulvestrant-dependent Interaction of ERα with CK8 and CK18 and Antiestrogen-induced Immobilization of ERα to the Nuclear Matrix and Receptor Degradation—Previous studies have suggested a role of two domains of ERα in ICI-induced receptor immobilization and degradation; that is, H12 and the F domain. Furthermore, Katzenellenbogen and coworkers (29Montano M.M. Ekena K. Krueger K.D. Keller A.L. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 230-242Crossref PubMed Scopus (85)
Referência(s)