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J. Biol. Chem., Vol. 279, Issue 29, 29944-29951, July 16, 2004

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An Hsp27-related, Dominant-negative-acting Intracellular Estradiol-binding Protein^*

Hong Chen, Martin Hewison, Bing Hu¶, Manju Sharma||, Zijie Sun||, and John S. Adams**

From the Division of Endocrinology, Diabetes, and Metabolism and the ^¶Department of Pathology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048, the Division of Medical Sciences, the University of Birmingham, Queen Elizabeth Medical Center, Birmingham, B15 2TH, United Kingdom, and the ^||Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305

New World primates (NWPs) exhibit a compensated form of resistance to gonadal steroid hormones. We demonstrated recently that estrogen resistance in NWP cells was associated with the overexpression of two proteins, a nonreceptor-related, dominant-negative-acting estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol-binding protein (IEBP). Based on the N-terminal sequences of tryptic fragments of IEBP isolated from a 17-estradiol (E₂) affinity column we cloned a full-length cDNA for IEBP from the estrogen-resistant NWP cell line, B95-8. Subsequent sequence analysis revealed 87% sequence identity between the deduced peptide for IEBP and human Hsp27. When hormone-responsive, wild-type Old World primate (OWP) cells were transiently transfected with IEBP cDNA, E₂-directed ERE reporter luciferase activity was reduced by 50% compared with vector only-transfected OWP cells (p < 0.0018). When IEBP and ERE-BP were cotransfected, ERE promoter-reporter activity was reduced by a further 60% (p < 0.0001). Electrophoresis mobility shift analyses showed that IEBP neither bound to ERE nor competed with the estrogen receptor (ER) for binding to ERE. However, there was evidence of protein-protein interaction of IEBP and ER; IEBP was coimmunoprecipitated with anti-ER antibody in wild-type cells stably transfected with IEBP. A specific interaction between ER and IEBP was confirmed in glutathione S-transferase pull-down and yeast two-hybrid assays. Data indicate that the Hsp27-related IEBP interacts with the ligand binding domain of the ER. In summary, by inhibiting the ER-E₂ interaction, IEBP acts to squelch ER-directed ERE-regulated transactivation and promote estrogen resistance in NWP cells.

Received for publication, February 6, 2004 , and in revised form, April 23, 2004.

^* This work was supported by National Institutes of Health Grant RO1DK55843 (to H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY518309.

^** To whom correspondence should be addressed: Division of Endocrinology, Diabetes, and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. B-131, Los Angeles, CA 90048. Tel.: 310-423-8970; Fax: 310-423-0440; E-mail: adamsj{at}cshs.org.

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This article has been cited by other articles:

				H. Chen, T. L. Clemens, M. Hewison, and J. S. Adams Estradiol and Tamoxifen Mediate Rescue of the Dominant-Negative Effects of Estrogen Response Element-Binding Protein in Vivo and in Vitro Endocrinology, May 1, 2009; 150(5): 2429 - 2435. [Abstract] [Full Text] [PDF]

				H. Chen, M. Hewison, and J. S. Adams Control of Estradiol-Directed Gene Transactivation by an Intracellular Estrogen-Binding Protein and an Estrogen Response Element-Binding Protein Mol. Endocrinol., March 1, 2008; 22(3): 559 - 569. [Abstract] [Full Text] [PDF]