Suppression of Estrogen Receptor-mediated Transcription and Cell Growth by Interaction with TR2 Orphan Receptor

Abstract

The transcriptional activity of the estrogen receptor (ER) is known to be highly modulated by the character and amount of coregulator proteins present in the cells. TR2 orphan receptor (TR2), a member of the nuclear receptor superfamily without identified ligands, is found to be expressed in the breast cancer cell lines and to function as a repressor to suppress ER-mediated transcriptional activity. Utilizing an interaction blocker, ER-6 (amino acids 312-340), responsible for TR2 interaction, the suppression of ER by TR2 could be reversed, suggesting that this suppression is conferred by the direct protein-protein interaction. Administration of antisense TR2, resulting in an enhancement of ER transcriptional activity in MCF7 cells, indicates that endogenous TR2 normally suppresses ER-mediated signaling. To gain insights into the molecular mechanism by which TR2 suppresses ER, we found that TR2 could interrupt ER DNA binding via formation of an ER-TR2 heterodimer that disrupted the ER homodimerization. The suppression of ER transcription by TR2 consequently caused the inhibition of estrogen-induced cell growth and G 1 /S transition in estrogen-dependent breast cancer cells. Taken together in addition to the potential roles in spermatogenesis and neurogenesis, our data provide a novel biological function of TR2 that may exert an important repressor in regulating ER activity in mammary glands.The human TR2 orphan receptor (TR2), 1 a member of the nuclear hormone receptor superfamily, was cloned from human testis and prostate cDNA libraries and has no identified ligand(s) (1, 2). TR2 is mapped to locate on chromosome 12q22 (3), which is known to be frequently deleted in various tumors, including testicular and ovarian germ cell tumors (4, 5). Four RNA isoforms, TR2-5, -7, -9, and -11, have been identified. Although TR2-11 encodes the full-length receptor, TR2-5, -7, and -9 encode truncated receptors with distinct deletions in the ligand binding domains (LBD) (1). TR2 has high homology with TR4, which places them in a unique subfamily within the nuclear hormone receptor superfamily (6). TR2 is evolutionarily conserved among species from primitive creatures to mammalians, including sea urchin, rainbow trout, axolotl, Xenopus, Drosophila, mouse, and human (1, 2, 7-11). The facts that TR2 is broadly expressed in many tissues throughout development starting at as early as mid-gestation stage (12-15) and that Drosophila with null mutations of DHR78 nuclear receptor, a homolog of human TR2, is lethal at the third-instar larval stage with severe defects in ecdysteroid-triggered metamorphosis (16), imply that the biological importance of TR2 is involved in the development process. With prominent expression throughout the active proliferating zones of the neural areas, the sensory nerve-targeted organs, and the testes during development, TR2 has been proposed to play an important role in the early development of the nervous system and the male reproductive system (12-15). Also, it has been shown that TR2 is primarily expresse...

Functional Domain and Motif Analyses of Androgen Receptor Coregulator ARA70 and Its Differential Expression in Prostate Cancer

“…Androgen-responsive LNCaP prostate cancer cells were maintained in RPMI 1640 containing penicillin (25 units/ml), streptomycin (25 g/ml), and 10% FCS. Transfections were performed using SuperFect reagent (Qiagen) according to the manufacturer's instructions or the calcium phosphate precipitation method, as previously described (17,47). Briefly, 4 ϫ 10 5 and 1 ϫ 10 5 cells were plated on 60-mm and 6-well dishes, respectively, 24 h before transfection, and the media were changed to DMEM with 5% charcoal dextran-stripped serum (CSS) 1 h before transfection.…”

Estrogen receptor corepressors -- a role in human breast cancer?

“…Other known or proposed mechanisms through which repressors could influence ERα activity involve inhibition of ERα dimerization (TR2, SHP) (Johansson et al 1999, Hu et al 2002 and DNA binding (SHP, TR2, p53) (Johansson et al 1999, Liu et al 2001, Hu et al 2002, effects on ERα stability (BRCA1, NEDD8) (Brzovic et al 2003, Fan et al 2003, sequestration of ERα away from its place of action (MTA1s) (Kumar et al 2002), or simply serving as a scaffold for the recruitment of a multi-protein complex (SHARP) (Shi et al 2001).…”

“…The orphan nuclear receptor SHP (short heterodimer partner) was originally isolated in a yeast two-hybrid screen using several conventional and orphan members of the receptor superfamily, including RAR and TR (Seol et al 1996), and was subsequently shown to interact with and repress ERα (Seol et al 1998, Johansson et al 2000. SHP is not the only orphan receptor implicated as a corepressor -TR2 (testicular receptor 2) (Hu et al 2002), DAX-1 (DSS-AHC critical region on the X, gene 1) and COUP-TF (chicken ovalbumin upstream promoter-transcription factor) (Klinge et al 1997) also modulate ERα actions. RIP140 (or nuclear receptor interacting protein 1, Nrip1) was originally identified as an ERα coactivator by expression cloning using the ERα AF-2 in the presence of estrogen (Cavailles et al 1995).…”

Structure-Function Analysis of the Estrogen Receptor α Corepressor Scaffold Attachment Factor-B1

“…Additionally, NCoR can also function by locking the central components of the transcription initiation machinery (TFIIB and TATA-binding protein-associated factors) into a nonfunctional complex or confirmation that is not favorable for transcription (25). In contrast to repressors containing independent and transferable repression domains, the repressor of estrogen receptor activity functions by competing with coactivators for ER␣ binding sites (26), and the testicular orphan nuclear re-ceptor 2 prevents ER␣ homodimerization and its subsequent interaction with DNA (27). Other proteins such as repressor of tamoxifen transcriptional activity may modulate ER␣ by regulating facets of mRNA processing or stability (28).…”