Ligand-dependent and -independent subcellular/subnuclear dynamics uncover new functional roles of ERRs in endocrine and metabolic regulation

NUCLEAR RECEPTORS

Estrogen receptor-related receptors (ERRs) are closely related to the estrogen receptors (ERs) in their protein structures and consist of 3 closely related isoforms: ERRα, ERRβ and ERRγ. However, ERRs do not bind to estrogens (Es) or any other known physiological ligands, and thus they are classified as “orphan nuclear receptors”. Studies on the expression pattern of ERRs in prostatic cells and prostate is scarce. As the first step towards a better understanding of the significance of ERRs in prostate, we investigated their expression profiles in human prostatic cell lines, two primary prostatic carcinoma xenografts, and prostatic tissues by RT-PCR, immunohistochemistry, and Western blot analysis. The results of RT-PCR showed that ERRα transcripts were widely detected in all tested cell lines and xenografts; ERRβ transcripts were either not detected or weakly expressed in prostatic cell lines and xenografts, while ERRγ transcripts were moderately expressed in C4-2B and PC-3 cells, and two xenografts. Similar expression patterns were also shown in human hyperplastic and neoplastic prostatic tissues. ERRγ immunohistochemistry showed that immunosignals were localized to the prostatic epithelial cells. We also compared mRNA expression of ERRs with that of ERs in prostatic cell lines. The results showed that ERRs and ERs were co-expressed in most cell lines and xenografts. Based on these observations, we speculate that the two members of nuclear receptor subfamily, ERRs and ERs, may control overlapping regulatory pathways in the prostatic cells.

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

The Therapeutic Potential of Nuclear Receptor Modulators for Treatment of Metabolic Disorders: PPARγ, RORs, and Rev-erbs

Nuclear Receptors

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, β, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRβ, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

BackgroundBreast cancer (BC) is highly heterogeneous with ~ 60–70% of estrogen receptor positive BC patient’s response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERRβ in breast cancer.MethodsEstrogen related receptor β (ERRβ) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERRβ is a direct target of ERα, we investigated the expression of ERRβ in short hairpin ribonucleic acid knockdown of ERα breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ERα by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERRβ in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERRβ with survival in breast cancer patients.ResultsTissue microarray (TMA) analysis showed that ERRβ is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER + ve breast tumors and cell lines showed a significant expression of ERRβ compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERRβ and it was ERα dependent. Mechanistic analyses indicate that ERα directly targets ERRβ through estrogen response element and ERRβ also mediates cell cycle regulation through p18, p21cip and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERRβ promoter activity in ectopically co-expressed ERα and ERRβ breast cancer cell lines. Fluorescence-activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERRβ overexpressed MCF7 cells. Furthermore, ERRβ expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERRβ in breast cancer cells which provide a potential avenue to target ERRβ signaling pathway in breast cancer.ConclusionOur results indicate that ERRβ is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERRβ could be therapeutic target for the treatment of breast cancer.

Retinoblastoma-binding protein 2 (Rbp2) was originally identified as a retinoblastoma protein (RB) pocket domain-binding protein. Although Rbp2 has been shown to interact with RB, p107, TATA-binding protein, and T-cell oncogene rhombotin-2, the physiological function of Rbp2 remains unclear. Here we demonstrate that Rbp2 not only binds to nuclear receptors (NRs) but also enhances the transcription mediated by them. Rbp2 interacts with the DNA-binding domains of NRs and potentiates NR-mediated transcription in an AF-2-dependent manner. Both the N-terminal and C-terminal domains of Rbp2 are critical for the transactivation activity of Rbp2 on NRs. The C terminus is the NR-interacting region. In addition, RB functions in maximizing the effect of Rbp2 on the transcription by NRs. These results suggest that Rbp2 is a coregulator of NRs and define a potential role for Rbp2 in NR-mediated transcription.

The functional interaction between the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivator PGC-1alpha is crucial for the normal physiology of PPARgamma and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPARgamma ligand-binding domain bound to rosiglitazone and to a large PGC-1alpha fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1alpha and the PPARgamma coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1alpha is capable of binding to PPARgamma. Our studies reveal that the strong interaction of PGC-1alpha and PPARgamma is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1alpha indicate that the first PGC-1alpha motif is necessary and sufficient for PGC-1alpha to coactivate PPARgamma in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPARgamma and PGC-1alpha in glucose homeostasis and adipocyte differentiation.

The oestrogen receptor recognizes an imperfectly palindromic response element through an alternative side-chain conformation.

Cholesterol, fatty acids, fat-soluble vitamins, and other lipids present in our diets are not only nutritionally important but serve as precursors for ligands that bind to receptors in the nucleus. To become biologically active, these lipids must first be absorbed by the intestine and transformed by metabolic enzymes before they are delivered to their sites of action in the body. Ultimately, the lipids must be eliminated to maintain a normal physiological state. The need to coordinate this entire lipid-based metabolic signaling cascade raises important questions regarding the mechanisms that govern these pathways. Specifically, what is the nature of communication between these bioactive lipids and their receptors, binding proteins, transporters, and metabolizing enzymes that links them physiologically and speaks to a higher level of metabolic control? Some general principles that govern the actions of this class of bioactive lipids and their nuclear receptors are considered here, and the scheme that emerges reveals a complex molecular script at work.

The orphan nuclear estrogen-related receptors (ERR) have homology to steroid hormone receptors. Unlike classical steroid hormone receptors, ERRs do not have known ligands but are transcriptionally activated upon binding to nuclear receptor coregulators such as PGC1α, PGC1β, and PPRC1. ERRs regulate energy metabolism and are accordingly expressed in tissues that require high energy production. ERRs recognize their own ERRE (estrogen-related receptor response element) gene promoters, but can also activate transcription via ERE (estrogen receptor response element) promoters, representing the ability of ERRs to cross participate in ERα regulatory pathways in the absence of estrogen. Of the three family members (α,β,γ), ERRα is the most highly expressed in breast tissue. ERRα expression was found to be increased in ERα negative breast cancers and associated with poor prognoses. ERRα and its coactivators can increase the expression of a critical mediator of breast tumor angiogenesis and metastasis, VEGFA. We have previously shown that VEGFA is regulated by insulin and IGF receptor activation and hypothesized that ERRα may be a mediator of this finding. We therefore explored whether ERRα and its coactivators are necessary for IGF-I/insulin dependent regulation of VEGFA and cell migration. ERRα was expressed in breast cancer cell lines, but no correlation was observed with breast cancer subtypes. Reducing endogenous ERRα levels by shRNA reduced IGF-I dependent migration in MDA-MB-231 cells. These results were confirmed using an ERRα specific inverse agonist (XCT-790), which also inhibited MDA-MB-231 cell migration. Modulating ERRα mRNA levels via shRNA resulted in decreased VEGFA mRNA levels in MCF7L cells and increased VEGFA mRNA levels in MDA-MB-231 cells, while the inverse agonist XCT-790 caused an increase in VEGFA mRNA levels in both cell lines. Reducing ERRa levels did not affect the expression of the IGF1R, InsR, IRS-1, IRS-2, PI-3K-Akt-mTOR dependent signaling pathway. Neither ERRα protein nor mRNA levels were modulated by IGF-I or insulin. We conclude that ERRα is important for mediating IGF-I dependent migration, but have shown that ERRα is not directly regulated by the IGF signaling. We will next explore the role of ERRα's coregulators (PGC1α, PGC1β, and PPRC1) in the regulation of VEGFA by the IGF/insulin pathway given their central role in regulating ERRα's function. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-09.

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.

Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

The estrogen-related receptors (ERRs) ┙, ┚ and ┛ comprise the NR3B orphan subgroup within the nuclear receptor superfamily. Although the ERRs were identified based on their sequence homology to estrogen receptor alpha (ER┙), they do not bind estrogen or any other natural hormones. Recent studies have defined the roles of ERRs in the regulation of target genes at the transcriptional level as well as their participation in a broad range of physiological functions such as energy metabolism and growth progression. The expression of ERRs has been shown to be up-regulated in advanced breast cancer cells and is considered to be a negative prognostic marker for the diagnosis of the disease. This review will cover what is currently known in regards to the gene structure of ERRs in addition to their regulation, function and relationship to breast cancer. Breast cancer is a complicated disease with 200,000 women diagnosed in the United States each year. There are many factors that influence breast cancer development and progression with hormone and nuclear receptors playing critical roles. Several reviews have been written on the emerging roles of estrogen and nuclear receptors in breast cancer (reviews (Conzen 2008; Hayashi, Niwa et al. 2009; Riggins, Mazzotta et al. 2010)). In this review we will focus on the estrogen-related receptor alpha, beta and gamma (ERR┙, ┚ and ┛). ERR┙ and ERR┚ were the first orphan nuclear receptors to be cloned in the late 1980’s (Giguere, Yang et al. 1988) with ERR┛ following 10 years later (Eudy, Yao et al. 1998; Hong, Yang et al. 1999; Heard, Norby et al. 2000). Although these receptors were cloned many years ago based on their sequence homology at the DNA binding domain to estrogen receptor alpha (ER┙), their biological relevance (s) has only recently been uncovered (Giguere 2008; Villena and Kralli 2008) along with potential roles in cancer, and more specifically breast cancer.