Estrogen receptor beta (ER beta) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ER beta and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ER alpha. Mouse ER beta bind to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner. Scatchard analysis indicates that mER beta has slightly lower affinity for E2 [dissociation constant (Kd) = 0.5 nM] when compared with mER alpha (Kd = 0.2 nM). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ER alpha on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mER beta. Cotransfection of mER beta and H-RasV12 causes enhanced activation in the presence of E2. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mER beta is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mER beta transactivation both in the absence and presence of E2, and in vitro interaction between SRC-1 and the ER beta ligand-binding domain is enhanced by E2. Moreover, the ligand-independent stimulatory effect of SRC-1 on ER beta transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ER beta shares many of the functional characteristics of ER alpha, the molecular mechanisms regulating the transcriptional activity of mER beta may be distinct from those of ER alpha.

The estrogen receptors (ERs) alpha and beta possess a constitutive N-terminal activation function (AF-1) whose activity can be modulated by kinase signalling pathways. We demonstrate here that phosphorylation of AF-1 by MAP kinase (MAPK) leads to the recruitment of steroid receptor coactivator-1 (SRC-1) by ER beta in vitro. Enhancement of the interaction between SRC-1 and ER beta AF-1 is also observed in vivo in cells either treated with EGF or expressing activated Ras. Two serine residues in ER beta AF-1, of which one is contained within a motif present in other steroid receptors, are critical for physical interaction with SRC-1 and transcriptional activation. Our results establish a role for nuclear receptor phosphorylation in the recruitment of SRC-1 and provide a molecular basis for ligand-independent activation by ER beta via the MAPK pathway.

The orphan nuclear receptor ERR␤ is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR␤ results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR␤ and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DEStreated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development. Received December 12, 2000; revised version accepted February 7, 2001. Nuclear receptors constitute a large family of transcription factors that mediate responses to small lipophilic hormones and play essential roles in embryonic development and maintenance of homeostasis in adult animals (Mangelsdorf et al. 1995). Orphan nuclear receptors are members of the nuclear receptor family that lack identified ligands (Giguère 1999). Recent studies have identified several natural and synthetic ligands for a number of orphan nuclear receptors, which led to the discovery of new hormone-response systems implicated in the control of cell fate, organogenesis, and basic metabolic functions Kliewer et al. 1999). While the estrogen-receptor-related receptor (ERR) ␣ (NR3B1) and ERR␤ (NR3B2) were the first orphan nuclear receptors identified more than a decade ago (Giguère et al. 1988), the identification of natural and synthetic ERR ligands has remained elusive. Despite being closely related to the estrogen receptors, ERRs are not activated by natural estrogens (Giguère et al. 1988). In contrast, members of the ERR family, now known to contain a third member referred to as ERR␥ (NR3B3) (Eudy et al. 1998), display various levels of constitutive activity and can interact with coactivators in the absence of ligand Vanacker et al. 1999;Xie et al. 1999).Molecular and genetic studies have shown that ERR␤ plays an important role in early placentation. ERR␤ is expressed in undifferentiated trophoblast stem cell lines (Tanaka et al. 1998) and in a subset of cells in extraembryonic ectoderm destined to make up the chorion (Pettersson et al. 1996;Luo et al. 1997). Genetic ablation of ERR␤ in the mouse results in abnormal chorion formation followed by failure of diploid trophoblast self-renewal and an increase in trophoblast giant cells (Luo et al. 1997). Thus, ERR␤ is essential for normal placental formation, and pharmacological modulation of its activity could influence reproductive function.Diethylstilbestrol (DES) is a potent synthetic estrogen that was used clinically for the prevention of spontaneous abortions from the 1940s to 1971 (Smith 1948). The mechanism of action and factual effectiveness of DES ...

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.

The orphan nuclear estrogen-related receptor ␣ (ERR␣) and transcriptional cofactor peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) are involved in the regulation of energy metabolism. Recently, extensive cross-talk between PGC-1␣ and ERR␣ has been demonstrated. The presence of PGC-1␣ is associated with an elevated expression of ERR␣, and the two proteins can influence the transcriptional activities of one another. Using a candidate gene approach to detect regulatory variants within genes encoding nuclear receptors, we have identified a 23-bp sequence (ESRRA23) containing two nuclear receptor recognition half-site motifs that is present in 1-4 copies within the promoter of the human ESRRA gene encoding ERR␣. The ES-RRA23 sequence contains a functional ERR response element that is specifically bound by ERR␣, and chromatin immunoprecipitation shows that endogenous ERR␣ occupies its own promoter in vivo. Strikingly, introduction of PGC-1␣ in HeLa cells by transient transfection induces the activity of the ESRRA promoter in a manner that is dependent on the presence of the ES-RRA23 element and on its dosage. Coexpression of ERR␣ and PGC-1␣ results in a synergistic activation of the ESRRA promoter. In experiments using ERR␣ null fibroblasts, the ability of PGC-1␣ to stimulate the ESRRA promoter is considerably reduced but can be restored by addition of ERR␣. Taken together, these results demonstrate that an interdependent ERR␣/PGC-1␣-based transcriptional pathway targets the ESRRA23 element to dictate the level of ERR␣ expression. This study further suggests that this regulatory polymorphism may provide differential responses to ERR␣/PGC-1␣-mediated metabolic cues in the human population.Nuclear hormone receptors are transcription factors that control essential developmental and physiological pathways (1). Although the transcriptional activity of nuclear receptors is primarily regulated by specific ligands, several members of the superfamily of nuclear receptors have no known natural ligands and are therefore referred to as orphan receptors (2). Estrogen-related receptor ␣ (ERR␣ 1 ; NR3B1) was the first orphan nuclear receptor to be identified on the basis of its similarity with estrogen receptor ␣ (ER␣; NR3A1) (3). Phylogenic tree reconstruction confirmed that ERR␣ belongs to the subgroup of receptors for steroid hormones (4), and ERR␣ was subsequently shown to share both structural and functional attributes with the ERs including binding to synthetic estrogenic ligands (reviewed in Ref. 5). ERR␣ also recognizes estrogen response elements (EREs), but characterization of its DNA binding properties demonstrated a preference for sites composed of a single half-site preceded by three nucleotides with the consensus sequence TNAAGGTCA, referred to as an ERRE (6 -10). The transcriptional activity of ERR␣ is independent of exogenously added ligand, and its relative potency as a transcriptional activator appears to be cell context-and promoterdependent (3,8,(11)(12)(13)(14)(15). ERR␣ has also been describ...

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Estrogen receptor beta (ER beta) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ER beta and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ER alpha. Mouse ER beta bind to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E2)-dependent manner. Scatchard analysis indicates that mER beta has slightly lower affinity for E2 [dissociation constant (Kd) = 0.5 nM] when compared with mER alpha (Kd = 0.2 nM). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E2-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ER alpha on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mER beta. Cotransfection of mER beta and H-RasV12 causes enhanced activation in the presence of E2. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mER beta is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mER beta transactivation both in the absence and presence of E2, and in vitro interaction between SRC-1 and the ER beta ligand-binding domain is enhanced by E2. Moreover, the ligand-independent stimulatory effect of SRC-1 on ER beta transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ER beta shares many of the functional characteristics of ER alpha, the molecular mechanisms regulating the transcriptional activity of mER beta may be distinct from those of ER alpha.

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