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Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor α Are Required to Elicit Cellular Alterations

2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês

10.1074/jbc.m900365200

1083-351X

Stephanie L. Nott, Yanfang Huang, Xiaodong Li, Brian Fluharty, Xing Qiu, Wade V. Welshons, Shuyuan Yeh, Mesut Muyan,

Cytokine Signaling Pathways and Interactions

Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) α and β. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ERα through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ERα also modulates gene expression by interacting with transregulators bound to cognate DNA-regulatory elements, and this regulation is referred to as the ERE-independent signaling pathway. The relative importance of the ERE-independent pathway in E2-ERα signaling is unclear. To address this issue, we engineered an ERE-binding defective ERα mutant (ERαEBD) by changing residues in an α-helix of the protein involved in DNA binding to render the receptor functional only through the ERE-independent signaling pathway. Using recombinant adenovirus-infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that E2-ERαEBD modulated the expression of a subset of ERα-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ERαEBD did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ERα.ERαEBD in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ERα, on the other hand, effectively repressed growth in this cell line. Our findings suggest that genomic responses from the ERE-dependent signaling pathway are required for E2-ERα to induce alterations in cellular responses. Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) α and β. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ERα through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ERα also modulates gene expression by interacting with transregulators bound to cognate DNA-regulatory elements, and this regulation is referred to as the ERE-independent signaling pathway. The relative importance of the ERE-independent pathway in E2-ERα signaling is unclear. To address this issue, we engineered an ERE-binding defective ERα mutant (ERαEBD) by changing residues in an α-helix of the protein involved in DNA binding to render the receptor functional only through the ERE-independent signaling pathway. Using recombinant adenovirus-infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that E2-ERαEBD modulated the expression of a subset of ERα-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ERαEBD did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ERα.ERαEBD in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ERα, on the other hand, effectively repressed growth in this cell line. Our findings suggest that genomic responses from the ERE-dependent signaling pathway are required for E2-ERα to induce alterations in cellular responses. 17β-Estradiol (E2), 5The abbreviations used are: E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; LBD, ligand binding domain; DBD, DNA binding domain; TUNEL, terminal dUTP nick-end labeling; PAS, plasminogen activator system; FACS, fluorescence-activated cell sorter; ECM, extracellular matrix; uPA, urokinase-plasminogen activator; uPAR, uPA receptor; HA, hyaluronan; m.o.i., multiplicity of infection; CD-FBS, charcoal-dextran treated-fetal bovine serum; qPCR, quantitative PCR; EMSA, electrophoretic mobility shift assay; ICC, immunocytochemistry; WB, Western blot; ChIP, chromatin immunoprecipitation assay; V, vector; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. as the main circulating estrogen hormone, plays critical roles in the physiology and pathophysiology of many tissues (1Deroo B.J. Korach K.S. J. Clin. Investig. 2006; 116: 561-570Crossref PubMed Scopus (1009) Google Scholar, 2Huang J. Li X. Hilf R. Bambara R.A. Muyan M. Curr. Drug. Targets Immune Endocr. Metabol. Disord. 2005; 5: 379-396Crossref PubMed Scopus (23) Google Scholar). The effects of E2 are primarily mediated by estrogen receptor (ER) α and β (1Deroo B.J. Korach K.S. J. Clin. Investig. 2006; 116: 561-570Crossref PubMed Scopus (1009) Google Scholar, 2Huang J. Li X. Hilf R. Bambara R.A. Muyan M. Curr. Drug. Targets Immune Endocr. Metabol. Disord. 2005; 5: 379-396Crossref PubMed Scopus (23) Google Scholar). ERs display functionally distinct structural features. The amino terminus of ERα contains a ligand-independent transactivation function. The central region is the DNA binding domain (DBD). The flexible hinge domain contains a nuclear localization signal and links the DBD domain to the multifunctional carboxyl-terminal ligand binding (LBD) domain. The LBD is involved in ligand binding, dimerization, and ligand-dependent transactivation function. Following synthesis, ERα dimerizes and translocates to the nucleus independent of E2 (3Bai Y. Giguére V. Mol. Endocrinol. 2003; 17: 589-599Crossref PubMed Scopus (60) Google Scholar). Fractions of the ERα population also partition to the perimembrane, cytoplasm, and mitochondria (4Hammes S.R. Levin E.R. Endocr. Rev. 2007; 28: 726-741Crossref PubMed Scopus (413) Google Scholar). The binding of E2 to ERα leads to a major structural reorganization of the LBD that converts the inactive ERα to the functionally active form by generating surfaces that support protein-protein interactions (5Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engström O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2963) Google Scholar). The integration of E2-ERα signaling generated from various cellular locations is thought to be critical for the regulation of responsive gene expression involved in cellular proliferation, differentiation, motility, and death (4Hammes S.R. Levin E.R. Endocr. Rev. 2007; 28: 726-741Crossref PubMed Scopus (413) Google Scholar, 6Björnström L. Sjöberg M. Mol. Endocrinol. 2005; 19: 833-842Crossref PubMed Scopus (1043) Google Scholar). One of the primary nuclear E2-ERα signaling events involves the interaction of E2-ERα with specific DNA sequences, known as estrogen-responsive elements (EREs) (7Klinge C.M. Nucleic Acids Res. 2001; 29: 2905-2919Crossref PubMed Scopus (812) Google Scholar), of estrogen-responsive genes and subsequent regulation of transcription. This signaling route is referred to as the ERE-dependent signaling pathway (2Huang J. Li X. Hilf R. Bambara R.A. Muyan M. Curr. Drug. Targets Immune Endocr. Metabol. Disord. 2005; 5: 379-396Crossref PubMed Scopus (23) Google Scholar, 8Hall J.M. Couse J.F. Korach K.S. J. Biol. Chem. 2001; 276: 36869-36872Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). E2-ERα also regulates gene transcription by interacting with a transcription factor, for example SP1 (stimulatory protein 1) and AP1 (activator protein 1), bound to cognate DNA-responsive elements on the regulatory regions of responsive genes. This nuclear E2-ER signaling is called the ERE-independent signaling pathway (2Huang J. Li X. Hilf R. Bambara R.A. Muyan M. Curr. Drug. Targets Immune Endocr. Metabol. Disord. 2005; 5: 379-396Crossref PubMed Scopus (23) Google Scholar, 8Hall J.M. Couse J.F. Korach K.S. J. Biol. Chem. 2001; 276: 36869-36872Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). However, the importance of the ERE-independent pathway in E2-ERα signaling in physiology and its contribution to pathophysiology remain unclear. Studies showed that changing Glu and Gly residues to Ala in the DNA-binding helix of the DBD of mouse (9Jakacka M. Ito M. Weiss J. Chien P.Y. Gehm B.D. Jameson J.L. J. Biol. Chem. 2001; 276: 13615-13621Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) and human (10DeNardo D.G. Kim H.T. Hilsenbeck S. Cuba V. Tsimelzon A. Brown P.H. Mol. Endocrinol. 2005; 19: 362-378Crossref PubMed Scopus (90) Google Scholar) ERα generates a mutant receptor capable of mediating E2 signaling through the ERE-independent pathway. Analogous mutations in the DBD of the human ERβ (11Björnström L. Sjöberg M. J. Biol. Chem. 2002; 277: 48479-48483Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar) also render the receptor functional only in the ERE-independent signaling pathway. Studies with a knock-in mouse model provide compelling support for the importance of the ERE-independent pathway in the regulation of various tissue functions, albeit in a tissue-specific manner (13Jakacka M. Ito M. Martinson F. Ishikawa T. Lee E.J. Jameson J.L. Mol. Endocrinol. 2002; 16: 2188-2201Crossref PubMed Scopus (166) Google Scholar, 14Christian C.A. Glidewell-Kenney C. Jameson J.L. Moenter S.M. Endocrinology. 2008; 149: 5328-5334Crossref PubMed Scopus (69) Google Scholar, 15Glidewell-Kenney C. Weiss J. Hurley L.A. Levine J.E. Jameson J.L. Endocrinology. 2008; 149: 4168-4176Crossref PubMed Scopus (35) Google Scholar). In an attempt to correlate genomic responses from the ERE-independent signaling pathway to alterations in cellular phenotypes, we found that changing Glu and Gly residues at positions 203 and 204 to Ala in the DNA-binding helix of human ERα DBD (ERα203/4) reduces but does not prevent functional features of the parent ERα in the ERE-dependent signaling pathway. Based on these observations, we introduced additional mutations in the DBD of ERα203/4. The replacement of Arg at position 211 with Glu in ERα203/4 generated an ERE-binding defective ERα mutant (ERα203/4/11 or ERαEBD) that was functional exclusively at the ERE-independent signaling pathway. We then assessed the effects of ERαEBD on cellular responses to E2 in adenovirus-infected ER-negative MDA-MB-231 cells derived from breast adenocarcinoma, in which exogenously introduced ERα was shown to induce cellular responses (16Garcia M. Derocq D. Freiss G. Rochefort H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11538-11542Crossref PubMed Scopus (158) Google Scholar, 17Jiang S.Y. Jordan V.C. J. Natl. Cancer Inst. 1992; 84: 580-591Crossref PubMed Scopus (244) Google Scholar, 18Lazennec G. Katzenellenbogen B.S. Mol. Cell. Endocrinol. 1999; 149: 93-105Crossref PubMed Scopus (41) Google Scholar). Our results reveal that ERαEBD mediated gene expression identified by a global gene expression profiling approach and verified by qPCR was insufficient to alter the proliferation, death, or motility of cells in contrast to E2-ERα. E2-ERαEBD was also ineffective in altering the phenotypic characteristics of ER-negative osteosarcoma-derived U-2OS cells used as an estrogen target tissue model wherein E2-ER signaling induces genomic and cellular changes (19Monroe D.G. Getz B.J. Johnsen S.A. Riggs B.L. Khosla S. Spelsberg T.C. J. Cell. Biochem. 2003; 90: 315-326Crossref PubMed Scopus (134) Google Scholar, 20Kian Tee M. Rogatsky I. Tzagarakis-Foster C. Cvoro A. An J. Christy R.J. Yamamoto K.R. Leitman D.C. Mol. Biol. Cell. 2004; 15: 1262-1272Crossref PubMed Scopus (197) Google Scholar, 21Stossi F. Barnett D.H. Frasor J. Komm B. Lyttle C.R. Katzenellenbogen B.S. Endocrinology. 2004; 145: 3473-3486Crossref PubMed Scopus (194) Google Scholar). Our results suggest that genomic responses from ERE-independent signaling pathways can be dissociated from the induction of phenotypic alterations. These results further imply that the ERE-dependent pathway is a required signaling route for E2-ERα to induce cellular responses. Generation of DNA Binding Defective ERα Mutants—The human ERα cDNA encoding the 595-amino acid long ERα was described previously (22Muyan M. Yi P. Sathya G. Willmert L.J. Driscoll M.D. Hilf R. Bambara R.A. Mol. Cell. Endocrinol. 2001; 182: 249-263Crossref PubMed Scopus (14) Google Scholar, 23Yi P. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 1810-1827Crossref PubMed Scopus (40) Google Scholar). This ERα cDNA also contains sequences that encode an amino-terminal FLAG epitope (23Yi P. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 1810-1827Crossref PubMed Scopus (40) Google Scholar). For the engineering of an ERE-binding defective ERα mutant, we utilized an overlapping PCR with the ERα cDNA as the template. The ERα203/4 mutant was generated using primers that contain sequences for amino acid substitutions replacing glutamic acid and glycine at positions 203 and 204, respectively, with alanine residues in the first zinc finger of the DBD. In the generation of the ERα203/4/11, ERα203/4 was used as the PCR template using primers with sequences that replace arginine at position 211 with glutamic acid. Restriction and DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA) and Invitrogen. Cell Culture—Culturing of Chinese hamster ovary, HeLa, and MDA-MB-231 cells was described previously (22Muyan M. Yi P. Sathya G. Willmert L.J. Driscoll M.D. Hilf R. Bambara R.A. Mol. Cell. Endocrinol. 2001; 182: 249-263Crossref PubMed Scopus (14) Google Scholar, 23Yi P. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 1810-1827Crossref PubMed Scopus (40) Google Scholar). U-2OS cells derived from osteosarcoma were purchased from the ATCC (Manassas, VA). U-2OS cells were grown in McCoy's 5α medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Invitrogen). C4 and C4-12 cells were grown in Eagle's modified essential medium without phenol red containing 5% CD-FBS (Hyclone). In all experiments, medium was changed every 3rd day. Transient Transfections—Transient transfections for simulated ERE-dependent and ERE-independent pathways were accomplished as described previously (23Yi P. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 1810-1827Crossref PubMed Scopus (40) Google Scholar, 24Huang J. Li X. Yi P. Hilf R. Bambara R.A. Muyan M. Mol. Cell. Endocrinol. 2004; 218: 65-78Crossref PubMed Scopus (30) Google Scholar, 25Li X. Huang J. Yi P. Bambara R.A. Hilf R. Muyan M. Mol. Cell. Biol. 2004; 24: 7681-7694Crossref PubMed Scopus (113) Google Scholar). Transfected MDA-MB-231 cells were treated without or with 10-9m 17β-estradiol (E2) and/or 10-7m Imperial Chemical Industries 182,780 (ICI, Tocris Inc., Ballwin, MO) for 24 or 40 h to assess the effects of ligands on ER-mediated transcriptional responses from the ERE-dependent or ERE-independent signaling, respectively. Generation of Recombinant Adenoviruses—Recombinant adenovirus bearing none or an ERα cDNA was produced by using the AdEasy-XL adenoviral system (Stratagene, La Jolla, CA) as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar, 26Huang J. Li X. Maguire C.A. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2005; 19: 2696-2712Crossref PubMed Scopus (35) Google Scholar). The purified viruses were titered using an Adeno-X rapid titer kit (BD Biosciences) to determine the multiplicity of infection (m.o.i.). Immunocytochemistry (ICC), Western Blot (WB), and Electrophoretic Mobility Shift Assay (EMSA)—Transfected or infected cells in a time-dependent manner were processed for ICC, WB, and EMSA as described previously (22Muyan M. Yi P. Sathya G. Willmert L.J. Driscoll M.D. Hilf R. Bambara R.A. Mol. Cell. Endocrinol. 2001; 182: 249-263Crossref PubMed Scopus (14) Google Scholar, 23Yi P. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 1810-1827Crossref PubMed Scopus (40) Google Scholar, 27Yi P. Driscoll M.D. Huang J. Bhagat S. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2002; 16: 674-693PubMed Google Scholar). For WB, proteins were probed with the horseradish peroxidase-conjugated monoclonal FLAG antibody (M2-horseradish peroxidase; Sigma) using the ECL-Plus Western blotting kit (GE Healthcare). For EMSA, we used FLAG M2 antibody (Sigma). Images from WB and EMSA were analyzed and quantified by ImageQuant version 1.2 software (GE Healthcare). For ICC, an ERα-specific antibody (HC-20) (Santa Cruz Biotechnology, Santa Cruz, CA) was used to probe ER proteins and a fluorescein-conjugated secondary antibody (Santa Cruz Biotechnology) for visualization. In Situ E2 Binding Assay—To assess the synthesis and functionality of ERα species in transfected or infected cells, we used the in situ E2 binding assay as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar, 26Huang J. Li X. Maguire C.A. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2005; 19: 2696-2712Crossref PubMed Scopus (35) Google Scholar). In brief, transiently transfected or infected cells were incubated with 10-7m [2,4,6,7,16,17-3H]17β-estradiol (118 Ci/mmol; PerkinElmer Life Sciences) in the absence or presence of 10-6m ICI for 1 h. Cells were then washed extensively with phosphate-buffered saline and collected, and radioactivity was measured in a scintillation counter. Chromatin Immunoprecipitation Assay (ChIP)—ChIP assays in transiently transfected MDA-MB-231 cells were performed using FLAG-M2 antibody-conjugated agarose beads (Sigma) as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar, 26Huang J. Li X. Maguire C.A. Hilf R. Bambara R.A. Muyan M. Mol. Endocrinol. 2005; 19: 2696-2712Crossref PubMed Scopus (35) Google Scholar). The generation of a 366-bp PCR fragment indicates the specificity of PCRs. Endogenous Gene Expression—MDA-MB-231 cells (100,000 cells/well), plated in 6-well tissue culture plates in phenol red-free Dulbecco's modified Eagle's medium containing 10% CD-FBS for 24 h, were infected with recombinant adenoviruses in the absence of ligands for 48 h to allow the synthesis of receptor proteins to reach maximum and comparable levels. Cells were then treated without or with 10-9m E2 for 6 or 24 h to assess the effects of ERs on the expression of endogenous genes. At termination, cells were collected and subjected to total RNA extraction using the RNeasy mini kit (Qiagen, Valencia, CA). For quantitative PCR (qPCR), we used custom TaqMan low density arrays with proprietary primer and probe sequences (Applied Biosystems, Foster City, CA) as we recently described (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). All qPCRs were carried out at the Functional Genomic Center of the University of Rochester, Rochester, NY. The expression of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as control. The real time reverse transcription-PCR amplifications were accomplished using an ABI Prism 7900HT sequence detection system with a TaqMan low density array upgrade (Applied Biosystems). Relative quantification analysis was performed using the comparative CT method (28Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127126) Google Scholar). Cellular Proliferation—MDA-MB-231 cells (5,000 cells/well) plated in 24-well tissue culture plates in phenol red-free Dulbecco's modified Eagle's medium containing 10% CD-FBS for 24 h were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 and/or 10-7m ICI for different durations of time. Cells were collected and counted using a hemocytometer (Hausser Scientific, Horsham, PA) and/or an automated cell counter (Nexcelom Biosciences, Lawrence, MA) or subjected to MTT assay as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). For the effects of ERs on U-2OS cell proliferation, cells (2,500 cells/well) were plated in 24-well tissue culture plates, precoated with poly-d-lysine hydrobromide (Sigma), in McCoy's α-medium containing 10% FBS for 24 h. Cells were subsequently incubated with fresh McCoy's α-medium containing 10% CD-FBS for an additional 24 h. Cells were then infected with recombinant adenoviruses in the absence or presence of 10-9m E2 and/or 10-7m ICI for different durations of time. We used Ad5-ERα at 40 m.o.i. At this m.o.i., the recombinant adenovirus synthesizes a concentration of ERα that requires E2 for function. Ad5-ERαEBD was used at 50 m.o.i., which produced comparable levels of receptor proteins to that of ERα. In all infections, the total m.o.i. was adjusted to 50 m.o.i. by the parent virus Ad5. At the termination of an experiment, cells were subjected to proliferation assays. Cell Cycle Analysis—MDA-MB-231 cells (50,000 cells/well) in 6-well tissue culture plates were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 and/or 10-7m ICI for different durations. Similarly, U-2OS cells (50,000 cells/well) in poly-d-lysine hydrobromide (Sigma)-coated 6-well tissue culture plates were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 and/or 10-7m ICI for various durations. At the termination of an experiment, collected cells were processed for and subjected to a fluorescence-activated cell sorting (FACS) using EPICS Elite (Coulter Corp., Miami, FL) as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). Annexin V and TUNEL Assays—The Vybrant apoptosis assay kit (Invitrogen) was used to study mid-stages of apoptosis as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). In brief, cells (100,000 cells/well) were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 for different lengths of time. Cells were collected and subjected to annexin V assay according to the instructions of the manufacturer prior to FACS analysis. For TUNEL assay, cells (25,000 cells/well) plated in poly-d-lysine hydrobromide-coated 48-well tissue culture plates in phenol red-free Dulbecco's modified Eagle's medium containing 10% CD-FBS were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 for different lengths of time. Cells were then subjected to a terminal dUTP nick-end labeling (TUNEL) assay utilizing the DeadEnd Flurometric TUNEL System (Promega) according to the manufacturer's protocol. 4′, 6-Diamidino-2-phenylindole (Vector Laboratories) was used to stain cell nuclei. Stained cells were imaged. Wound-healing Assay—MDA-MB-231 cells (200,000 cells/well in 12-well tissue culture plates) were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 for 48 h, a duration that allowed the cells to reach near-confluence. A wound was generated with a 1-ml pipette tip. The closure of wound was then imaged every 24 h. Because of the irregular shape of the edges of a wound, five randomly selected cross-edges were used to obtain a mean gap measure for wound healing. Invasion Assay—Matrigel invasion chambers (BD Biosciences) were used for the invasion assay. MDA-MB-231 cells (100,000 cells/well) in 6-well tissue culture plates were infected with recombinant adenoviruses in the absence or presence of 10-9m E2 for 48 h. Cells were then collected, and equal numbers of cells from each treatment group were seeded into invasion chambers as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). Cells on the bottom of the chamber membrane as the invading cell population were stained with the Diff-Quik Stain Set (Dade Behring, Newark, DE), dried, and mounted onto a glass slide. Images were captured, and stained cells were counted from images. Microarray Analysis—To examine the effects of E2 on endogenous gene expression mediated by ERs, MDA-MB-231 cells were infected with recombinant adenoviruses in the absence of E2 for 48 h. Cells were then treated with 10-9m E2 for 6 h. At the termination of an experiment, total RNA was extracted using RNeasy mini kit (Qiagen) and processed for microarray analysis, which was carried out at the Functional Genomic Center of the University of Rochester, as described previously (12Li X. Nott S.L. Huang Y. Hilf R. Bambara R.A. Qiu X. Yakovlev A. Welle S. Muyan M. J. Mol. Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar). Affymetrix HG-U133 Plus 2.0 arrays were used as the microarray platform. Arrays were scanned with the GeneChip Scanner 3000 7G. GeneChip operating software (Affymetrix) was used for initial processing of the scanner data, including generation of cel files. Array normalization for the Affymetrix signal method (GeneChip operating software) involves multiplying raw signals by a scaling factor such that the trimmed mean (excluding highest and lowest 2%) of all expression scores is 500 arbitrary units for every array. Experimental sets for microarrays were replicated six independent times executed on different days. To increase accuracy of gene identification and reduce false discovery rate (29Dai M. Wang P. Jakupovic E. Watson S.J. Meng F. Bioinformatics. 2007; 23: 2185-2187Crossref PubMed Scopus (11) Google Scholar), we used reorganized and updated probe sets (30Gautier L. Møller M. Friis-Hansen L. Knudsen S. BMC Bioinformatics. 2004; 5: 111Crossref PubMed Scopus (100) Google Scholar, 31Dai M. Wang P. Boyd A.D. Kostov G. Athey B. Jones E.G. Bunney W.E. Myers R.M. Speed T.P. Akil H. Watson S.J. Meng F. Nucleic Acids Res. 2005; 33: e175Crossref PubMed Scopus (1404) Google Scholar, 32Harbig J. Sprinkle R. Enkemann S.A. Nucleic Acids Res. 2005; 33: e31Crossref PubMed Scopus (102) Google Scholar) that were based on the up-to-date genome, cDNA/EST clustering, and single nucleotide polymorphism information through web-based custom GeneChip library files (Chip definition files) in data analysis. Following UniGene transformation, data sets were subjected to N-statistic test (33Klebanov L. Gordon A. Xiao Y. Land H. Yakovlev A. Comput. Statist. Data Anal. 2006; 50: 3619-3628Crossref Scopus (24) Google Scholar) in conjunction with the step-down Westfall-Young procedure (34Westfall P.H. Young S. Resampling-based Multiple Testing. Wiley Interscience, New York1993: 36-75Google Scholar) controlling the family-wise error rate at a level of 0.05, which we reported here. Minimum information about a microarray experiment-compliant microarray data for the six independent replicate studies can be found at the Gene Expression Omnibus data base with an accession number GSE9761. Statistical Analysis—Results presented as the mean ± S.E. of at least three independent experiments were subjected to Student's t test for comparison of the means between two groups wherein p < 0.05 was considered significant. Generation of a Mutant Human ERα for the Selective Regulation of the ERE-independent DNA-dependent Signaling Pathway—The initial step in the ERE-dependent signaling pathway involves the interaction of the E2-ERα complex with EREs, which are permutations of a core palindromic sequence, 5′-GGTCAnnnTGACC-3′ (7Klinge C.M. Nucleic Acids Res. 2001; 29: 2905-2919Crossref PubMed Scopus (812) Google Scholar). The recognition of an ERE is mediated by two zinc-binding motifs in each DBD monomer of the ERα dimer that fold to form a single functional unit (35Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (600) Google Scholar, 36Luisi B.F. Schwabe J.W. Freedman L.P. Vitam. Horm. 1994; 49: 1-47Crossref PubMed Scopus (33) Google Scholar). 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Endocrinol. 2008; 40: 211-229Crossref PubMed Scopus (7) Google Scholar) showed that Glu and Gly residues are also important for binding to ERE, as changing these amino acids to Ala residues is sufficient to hinder the transregulatory capacity of the receptor from the ERE signaling pathway. To begin to address the importance of the ERE-independent pathway in physiology and pathophysiology of E2-ERα signaling, we also engineered a human ERα mutant by changing Glu203 and Gly204 to Ala residues (ERα203/4). We found in preliminary studies that ERα203/4 retains, albeit at reduced levels, biochemical and functional features of the parent ERα in systems emulating the ERE-dependent signaling pathway (see also Figs. 1 and 2).FIGURE 2Transcriptional