This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, C. C. Articles by Shapiro, D. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, C. C. Articles by Shapiro, D. J.

HMG-1 Stimulates Estrogen Response Element Binding by Estrogen Receptor from Stably Transfected HeLa Cells

Department of Biochemistry University of Illinois Urbana, Illinois 61801

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17ß-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (K_D) for 17ß-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar K_D values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the K_D of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (K_D of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17ß-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.

This article has been cited by other articles:

				S. C. Roemer, J. Adelman, M. E. A. Churchill, and D. P. Edwards Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding Nucleic Acids Res., June 1, 2008; 36(11): 3655 - 3666. [Abstract] [Full Text] [PDF]

				M. P. Fink Neuropeptide Modulators of High Mobility Group Box 1 Secretion as Potential Therapeutic Agents for Severe Sepsis Am. J. Pathol., May 1, 2008; 172(5): 1171 - 1173. [Abstract] [Full Text] [PDF]

				C. D. Curtis, V. S. Likhite, I. X. McLeod, J. R. Yates, and A. M. Nardulli Interaction of the Tumor Metastasis Suppressor Nonmetastatic Protein 23 Homologue H1 and Estrogen Receptor {alpha} Alters Estrogen-Responsive Gene Expression Cancer Res., November 1, 2007; 67(21): 10600 - 10607. [Abstract] [Full Text] [PDF]

				J. Cheng, D. V. Yu, J.-H. Zhou, and D. J. Shapiro Tamoxifen Induction of CCAAT Enhancer-binding Protein {alpha} Is Required for Tamoxifen-induced Apoptosis J. Biol. Chem., October 19, 2007; 282(42): 30535 - 30543. [Abstract] [Full Text] [PDF]

				J. Cheng, C. Zhang, and D. J. Shapiro A Functional Serine 118 Phosphorylation Site in Estrogen Receptor-{alpha} Is Required for Down-Regulation of Gene Expression by 17{beta}-Estradiol and 4-Hydroxytamoxifen Endocrinology, October 1, 2007; 148(10): 4634 - 4641. [Abstract] [Full Text] [PDF]

				S. Wang, C. Zhang, S. K. Nordeen, and D. J. Shapiro In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors {alpha} and beta Supports an Active Displacement Model for Coregulator Utilization J. Biol. Chem., February 2, 2007; 282(5): 2765 - 2775. [Abstract] [Full Text] [PDF]

				S. C. Roemer, D. C. Donham, L. Sherman, V. H. Pon, D. P. Edwards, and M. E. A. Churchill Structure of the Progesterone Receptor-Deoxyribonucleic Acid Complex: Novel Interactions Required for Binding to Half-Site Response Elements Mol. Endocrinol., December 1, 2006; 20(12): 3042 - 3052. [Abstract] [Full Text] [PDF]

				S. F. Eddy, S. Guo, E. G. Demicco, R. Romieu-Mourez, E. Landesman-Bollag, D. C. Seldin, and G. E. Sonenshein Inducible I{kappa}B Kinase/I{kappa}B Kinase {varepsilon} Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-{kappa}B Activation in Breast Cancer Cells Cancer Res., December 15, 2005; 65(24): 11375 - 11383. [Abstract] [Full Text] [PDF]

				D. Das, R. C. Peterson, and W. M. Scovell High Mobility Group B Proteins Facilitate Strong Estrogen Receptor Binding to Classical and Half-Site Estrogen Response Elements and Relax Binding Selectivity Mol. Endocrinol., November 1, 2004; 18(11): 2616 - 2632. [Abstract] [Full Text] [PDF]

				M. A. Loven, R. E. Davis, C. D. Curtis, N. Muster, J. R. Yates, and A. M. Nardulli A Novel Estrogen Receptor {alpha}-Associated Protein Alters Receptor-Deoxyribonucleic Acid Interactions and Represses Receptor-Mediated Transcription Mol. Endocrinol., November 1, 2004; 18(11): 2649 - 2659. [Abstract] [Full Text] [PDF]

				V. S. Melvin, C. Harrell, J. S. Adelman, W. L. Kraus, M. Churchill, and D. P. Edwards The Role of the C-terminal Extension (CTE) of the Estrogen Receptor {alpha} and {beta} DNA Binding Domain in DNA Binding and Interaction with HMGB J. Biol. Chem., April 9, 2004; 279(15): 14763 - 14771. [Abstract] [Full Text] [PDF]

				A. J. Krieg, S. A. Krieg, B. S. Ahn, and D. J. Shapiro Interplay between Estrogen Response Element Sequence and Ligands Controls in Vivo Binding of Estrogen Receptor to Regulated Genes J. Biol. Chem., February 6, 2004; 279(6): 5025 - 5034. [Abstract] [Full Text] [PDF]

				A. L. Amir, M. Barua, N. C. McKnight, S. Cheng, X. Yuan, and S. P. Balk A Direct {beta}-Catenin-independent Interaction between Androgen Receptor and T Cell Factor 4 J. Biol. Chem., August 15, 2003; 278(33): 30828 - 30834. [Abstract] [Full Text] [PDF]

				M. A. Loven, N. Muster, J. R. Yates, and A. M. Nardulli A Novel Estrogen Receptor {alpha}-Associated Protein, Template-Activating Factor I{beta}, Inhibits Acetylation and Transactivation Mol. Endocrinol., January 1, 2003; 17(1): 67 - 78. [Abstract] [Full Text] [PDF]

				M. Obrero, D. V. Yu, and D. J. Shapiro Estrogen Receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced Programmed Cell Death J. Biol. Chem., November 15, 2002; 277(47): 45695 - 45703. [Abstract] [Full Text] [PDF]

				V. S. Melvin, S. C. Roemer, M. E. A. Churchill, and D. P. Edwards The C-terminal Extension (CTE) of the Nuclear Hormone Receptor DNA Binding Domain Determines Interactions and Functional Response to the HMGB-1/-2 Co-regulatory Proteins J. Biol. Chem., July 5, 2002; 277(28): 25115 - 25124. [Abstract] [Full Text] [PDF]

				X. Yuan, M. L. Lu, T. Li, and S. P. Balk SRY Interacts with and Negatively Regulates Androgen Receptor Transcriptional Activity J. Biol. Chem., November 30, 2001; 276(49): 46647 - 46654. [Abstract] [Full Text] [PDF]

				H. WANG, H. YANG, C. J. CZURA, A. E. SAMA, and K. J. TRACEY HMGB1 as a Late Mediator of Lethal Systemic Inflammation Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1768 - 1773. [Full Text] [PDF]

				S. A. Krieg, A. J. Krieg, and D. J. Shapiro A Unique Downstream Estrogen Responsive Unit Mediates Estrogen Induction of Proteinase Inhibitor-9, a Cellular Inhibitor of IL-1{beta}- Converting Enzyme (Caspase 1) Mol. Endocrinol., November 1, 2001; 15(11): 1971 - 1982. [Abstract] [Full Text] [PDF]

				C. J. Czura, Haichao Wang, and K. J. Tracey Dual roles for HMGB1: DNA binding and cytokine Innate Immunity, August 1, 2001; 7(4): 315 - 321. [Abstract] [PDF]

				S. Oesterreich, P. Zhang, R. L. Guler, X. Sun, E. M. Curran, W. V. Welshons, C. K. Osborne, and A. V. Lee Re-expression of Estrogen Receptor {alpha} in Estrogen Receptor {alpha}-negative MCF-7 Cells Restores both Estrogen and Insulin-like Growth Factor-mediated Signaling and Growth Cancer Res., August 1, 2001; 61(15): 5771 - 5777. [Abstract] [Full Text] [PDF]

				C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]

				L. R. Drew, D. C. Tang, P. E. Berg, and G. P. Rodgers The role of trans-acting factors and DNA-bending in the silencing of human {beta}-globin gene expression Nucleic Acids Res., July 15, 2000; 28(14): 2823 - 2830. [Abstract] [Full Text] [PDF]

				C. Mao and D. J. Shapiro A Histone Deacetylase Inhibitor Potentiates Estrogen Receptor Activation of a Stably Integrated Vitellogenin Promoter in HepG2 Cells Endocrinology, July 1, 2000; 141(7): 2361 - 2369. [Abstract] [Full Text] [PDF]

				K. B. Ellwood, Y.-M. Yen, R. C. Johnson, and M. Carey Mechanism for Specificity by HMG-1 in Enhanceosome Assembly Mol. Cell. Biol., June 15, 2000; 20(12): 4359 - 4370. [Abstract] [Full Text]

				D. Knutti, A. Kaul, and A. Kralli A Tissue-Specific Coactivator of Steroid Receptors, Identified in a Functional Genetic Screen Mol. Cell. Biol., April 1, 2000; 20(7): 2411 - 2422. [Abstract] [Full Text]

				H. Kanamori, S. Krieg, C. Mao, V. A. Di Pippo, S. Wang, D. A. Zajchowski, and D. J. Shapiro Proteinase Inhibitor 9, an Inhibitor of Granzyme B-mediated Apoptosis, Is a Primary Estrogen-inducible Gene in Human Liver Cells J. Biol. Chem., February 25, 2000; 275(8): 5867 - 5873. [Abstract] [Full Text] [PDF]

				C. C. Zhang and D. J. Shapiro Activation of the p38 Mitogen-activated Protein Kinase Pathway by Estrogen or by 4-Hydroxytamoxifen Is Coupled to Estrogen Receptor-induced Apoptosis J. Biol. Chem., January 7, 2000; 275(1): 479 - 486. [Abstract] [Full Text] [PDF]

				Q. He, C. H. Liang, and S. J. Lippard Steroid hormones induce HMG1 overexpression and sensitize breast cancer cells to cisplatin and carboplatin PNAS, May 23, 2000; 97(11): 5768 - 5772. [Abstract] [Full Text] [PDF]