Estrogen-related Receptor α1 Actively Antagonizes Estrogen Receptor-regulated Transcription in MCF-7 Mammary Cells

Abstract

The estrogen-related receptor ␣ (ERR␣) is an orphan member of the nuclear receptor superfamily. We show that the major isoform of the human ERR␣ gene, ERR␣1, can sequence-specifically bind a consensus palindromic estrogen response element (ERE) and directly compete with estrogen receptor ␣ (ER␣) for binding. ERR␣1 activates or represses ERE-regulated transcription in a cell type-dependent manner, repressing in ER-positive MCF-7 cells while activating in ER-negative HeLa cells. Thus, ERR␣1 can function both as a modulator of estrogen responsiveness and as an estrogen-independent activator. Repression likely occurs in the absence of exogenous ligand since charcoal treatment of the serum had no effect on silencing activity. Mutational analysis revealed that repression is not simply the result of competition between ER␣ and ERR␣1 for binding to the DNA. Rather, it also requires the presence of sequences within the carboxyl-terminal E/F domain of ERR␣1. Thus, ERR␣1 can function as either an active repressor or a constitutive activator of ERE-dependent transcription. We hypothesize that ERR␣1 can play a critical role in the etiology of some breast cancers, thereby providing a novel therapeutic target in their treatment. The nuclear receptor (NR)1 superfamily is comprised of hundreds of transcription factors that regulate a vast array of genes and physiological responses (1-9). Most nuclear receptors share a similar structural organization (Fig. 1A). The amino-terminal A/B domain can function as a hormone-independent activator of transcription. The highly conserved C domain contains the DNA binding domain (DBD) that confers sequence-specific DNA binding activity. A hinge region, called the D domain, bridges the C domain with the carboxyl-terminal E/F domain that includes the receptor-specific ligand binding domain (LBD) of the protein. The binding of appropriate ligands results in conformation changes leading to alterations in the transcriptional properties of the receptor, including the exposure of a transcriptional activation region within the carboxyl end. Although many nuclear receptor superfamily members bind known ligands (e.g., steroids, retinoids, thyroid hormones), some, termed orphan receptors, share significant sequence similarity in their LBDs with their ligand binding family members but lack as-yet known naturally occurring ligands (7-10).Among the first orphan receptors identified were the estrogen-related receptors ERR␣ and ERR␤ (officially named NR3B1 and NR3B2, respectively) (10). They were cloned by low stringency screening of cDNA libraries with probes corresponding to the DBD of estrogen receptor ␣ (ER␣) (10). Subsequently, ERR␣1 was identified as the major isoform present in HeLa cells (Fig. 1A) (11, 12). The DBD of human ERR␣1 shares 70% amino acid similarity with the DBD of human ER␣; the LBD shares 35% amino acid identity. A third member of the ERR family, ERR␥ (NR3B3), has also been identified (13-15). These three ERRs are closely related by sequence similarity but encoded by different genes.De...

Estrogen Induces Estrogen-related Receptor α Gene Expression and Chromatin Structural Changes in Estrogen Receptor (ER)-positive and ER-negative Breast Cancer Cells

“…Because its function is generally constitutive, ERR␣ could potentially induce ER␣ target genes in hormonally responsive cancers in an estrogen-independent manner. In addition, ERR␣ and ER␣ may antagonize each other's function on certain promoters (40,57). The correlation between overexpression of ERR␣ and CYP19A1 (49), a downstream target of ERR␣, in breast cancer cells suggests that ERR␣ may contribute to increased local production of estrogens in mammary glands.…”

Estrogen-Related Receptor α1 Transcriptional Activities Are Regulated in Part via the ErbB2/HER2 Signaling Pathway

“…2A), which served as an external control for experimental conditions, the physiologic state of the cells, and non -specific effects on the basal transcriptional machinery. The effect and specificity of ERRa1 was evaluated by cotransfecting the cells with a plasmid expressing wild-type ERRa1, mutant ERRa1 L413A/L418A , a variant defective in the carboxyl-terminal inverted LxLxxL motif that serves as a coactivator docking site (8), or their parental empty vector. The cells were also cotransfected with a plasmid that expressed GRIP1, a member of the p160 family of coactivators, or its empty parental plasmid.…”

“…The effect of ERRa1 binding to a transcriptional response element can be either negative or positive depending on the specific cell type (8) and promoter context (24). For example, ERRa1 downmodulates E 2 -induced transcription in ERa-positive human mammary carcinoma MCF-7 cells by an active mechanism (8), yet activates gene transcription in ERa-negative human mammary carcinoma SK-BR-3 cells (19) and a variety of other cell lines, including human cervical carcinoma HeLa cells (8), human endometrial RL95-2 cells (4), human embryonic kidney 293 cells (21), and rat ROS 17/2.8 osteosarcoma cells (21).…”

“…They do not bind naturally occurring estrogens, but share other biochemical activities with ERs, including binding to estrogen response elements (ERE; refs. [4][5][6][7][8]. ERRs also bind to extended nuclear receptor half-site sequences resembling 5 ¶-TNAAGGTCA-3 ¶, referred to as ERR response elements (ERRE; refs.…”

“…Thus, by analogy with ERa, we hypothesized that signaling via ErbB2 leads to phosphorylation of ERRa1, thereby modulating its activities. Findings in support of this hypothesis include the following: (a) ERRa1 can exist as a phosphoprotein (9); (b) human breast tumors that express high lev els of ErbB2 mRNA also frequently express high levels of ERRa mRNA (11); and (c) SK-BR-3 cells, in which ERRa1 functions as a constitutive activator (19), contain 2 orders of magnitude more ErbB2 mRNA than MCF-7 cells (33) in which it functions as a down-modulator of transcription (8).…”