Orphan Nuclear Receptor ERRα Controls Macrophage Metabolic Signaling and A20 Expression to Negatively Regulate TLR-Induced Inflammation.

The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming.

Multifaceted Transcriptional Network of Estrogen-Related Receptor Alpha in Health and Disease.

Estrogen-related receptors (ERRα, ß and γ in mammals) are orphan members of the nuclear receptor superfamily acting as transcription factors. ERRs are expressed in several cell types and they display various functions in normal and pathological contexts. Amongst others, they are notably involved in bone homeostasis, energy metabolism and cancer progression. In contrast to other nuclear receptors, the activities of the ERRs are apparently not controlled by a natural ligand but they rely on other means such as the availability of transcriptional co-regulators. Here we focus on ERRα and review the variety of co-regulators that have been identified by various means for this receptor and their reported target genes. ERRα cooperates with distinct co-regulators to control the expression of distinct sets of target genes. This exemplifies the combinatorial specificity of transcriptional regulation that induces discrete cellular phenotypes depending on the selected coregulator. We finally propose an integrated view of the ERRα transcriptional network.

Regulation of the expression of the estrogen related receptors (ERRs).

Estrogen related receptors (ERRα, ß and γ in mammals) are orphan members of the nuclear receptor superfamily acting as transcription factors. ERRs are expressed in several tissues and cells and they display various physiological and pathological functions, controlling, amongst others and depending on the receptor, bone homeostasis, energy metabolism, embryonic stem cell pluripotency, and cancer progression. In contrast to classical nuclear receptors, the activities of the ERRs are not controlled by a natural ligand. Regulation of their activities thus rely on other means such as post-translational modification or availability of transcriptional co-regulators. In addition, regulation of their mere expression under given physiological or pathological conditions is a particularly important level of control. Here we discuss the mechanisms involved in the regulation of ERRs expression and the reported means to impact on it using pharmacological approaches.

ERRα induces H3K9 demethylation by LSD1 to promote cell invasion.

Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.

The estrogen-related receptors (ERRs): potential targets against bone loss.

Bone loss and the resulting skeletal fragility is induced by several pathological or natural conditions, the most prominent of which being aging as well as the decreased levels of circulating estrogens in post-menopause females. To date, most treatments against bone loss aim at preventing excess bone resorption. We here summarize data indicating that the estrogen-related receptors (ERRs) α and γ prevent bone formation. Inhibiting these receptors may thus constitute an anabolic approach by increasing bone formation.

Estrogen-related receptor α decreases RHOA stability to induce orientated cell migration.

Several physiopathological processes require orientated cellular migration. This phenomenon highly depends on members of the RHO family of GTPases. Both excessive and deficient RHO activity impair directional migration. A tight control is thus exerted on these proteins through the regulation of their activation and of their stability. Here we show that the estrogen-related receptor α (ERRα) directly activates the expression of TNFAIP1, the product of which [BTB/POZ domain-containing adapter for Cullin3-mediated RhoA degradation 2 (BACURD2)] regulates RHOA protein turnover. Inactivation of the receptor leads to enhanced RHOA stability and activation. This results in cell disorientation, increased actin network, and inability to form a lamellipodium at the migration edge. As a consequence, directional migration, but not cell motility per se, is impaired in the absence of the receptor, under pathological as well as physiological conditions. Altogether, our results show that the control exerted by ERRα on RHOA stability is required for directional migration.

ERRα augments HIF-1 signalling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells.

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up-regulation of hypoxia-inducible factor 1 (HIF-1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen-related receptor-α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα-over-expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF-1α protein expression and HIF-1 signalling. These effects could also be observed in ERRα-over-expressing cells grown under normoxia, suggesting that ERRα could function to pre-adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF-1α via its AF-2 domain. A ubiquitination assay showed that this ERRα-HIF-1α interaction could inhibit ubiquitination of HIF-1α and thus reduce its degradation. Such ERRα-HIF-1α interaction could be attenuated by XCT790, an ERRα-specific inverse agonist, resulting in reduced HIF-1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF-1α, suggesting ERRα as a potential therapeutic target for cancer treatment.

ERRα: unraveling its role as a key player in cell migration.

Cell migration is essential throughout the life of multicellular organisms, and largely depends on the spatial and temporal regulation of cytoskeletal dynamics, cell adhesion and signal transduction. Interestingly, Estrogen-related receptor alpha (ERRα) has been identified as a major regulator of cell migration in both physiological and pathological conditions. ERRα is an orphan member of the nuclear hormone receptor superfamily of transcription factors and displays many biological functions. ERRα is a global regulator of energy metabolism, and it is also highly involved in bone homeostasis, development, differentiation, immunity and cancer progression. Importantly, in some instances, the regulation of these biological processes relies on the ability to orchestrate cell movements. Therefore, this review describes how ERRα-mediated cell migration contributes not only to tissue homeostasis but also to tumorigenesis and metastasis, and highlights the molecular and cellular mechanisms by which ERRα finely controls the cell migratory potential.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes.

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Computational identification of new potential transcriptional partners of ERRα in breast cancer cells: specific partners for specific targets.

Estrogen related receptors are orphan members of the nuclear receptor superfamily acting as transcription factors (TFs). In contrast to classical nuclear receptors, the activities of the ERRs are not controlled by a natural ligand. Regulation of their activities thus relies on availability of transcriptional co-regulators. In this paper, we focus on ERRα, whose involvement in cancer progression has been broadly demonstrated. We propose a new approach to identify potential co-activators, starting from previously identified ERRα-activated genes in a breast cancer (BC) cell line. Considering mRNA gene expression from two sets of human BC cells as major endpoint, we used sparse partial least squares modeling to uncover new transcriptional regulators associated with ERRα. Among them, DDX21, MYBBP1A, NFKB1, and SETD7 are functionally relevant in MDA-MB-231 cells, specifically activating the expression of subsets of ERRα-activated genes. We studied SET7 in more details and showed its co-localization with ERRα and its ERRα-dependent transcriptional and phenotypic effects. Our results thus demonstrate the ability of a modeling approach to identify new transcriptional partners from gene expression. Finally, experimental results show that ERRα cooperates with distinct co-regulators to control the expression of distinct sets of target genes, thus reinforcing the combinatorial specificity of transcription.

ERRα coordinates actin and focal adhesion dynamics.

Cell migration depends on the dynamic organisation of the actin cytoskeleton and assembly and disassembly of focal adhesions (FAs). However, the precise mechanisms coordinating these processes remain poorly understood. We previously identified the oestrogen-related receptor α (ERRα) as a major regulator of cell migration. Here, we show that loss of ERRα leads to abnormal accumulation of actin filaments that is associated with an increased level of inactive form of the actin-depolymerising factor cofilin. We further show that ERRα depletion decreases cell adhesion and results in defective FA formation and turnover. Interestingly, specific inhibition of the RhoA-ROCK-LIMK-cofilin pathway rescues the actin polymerisation defects resulting from ERRα silencing, but not cell adhesion. Instead, we found that MAP4K4 is a direct target of ERRα and down-regulation of its activity rescues cell adhesion and FA formation in the ERRα-depleted cells. Altogether, our results highlight a crucial role of ERRα in coordinating the dynamic of actin network and FAs through the independent regulation of the RhoA and MAP4K4 pathways.

MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

Thyroid hormone receptor beta (TRbeta) and liver X receptor (LXR) regulate carbohydrate-response element-binding protein (ChREBP) expression in a tissue-selective manner.

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRbeta but not by TRalpha in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta(-/-) mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRbeta signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems.

Conserved properties of a urochordate estrogen receptor-related receptor (ERR) with mammalian ERRalpha.

Estrogen receptor-related receptors (ERRs) were the first orphan nuclear receptors identified on the basis of their sequence similarity to the estrogen receptors. Although unique ERRs were found in some marine invertebrates, the molecular functions of these receptors are not well understood. In the present study, we identified three transcript variants of the tunicate Halocynthia roretzi ERR (Hr-ERR), varying in their 3' untranslated regions, and putatively encoding a unique receptor deriving from an ancestor protein common to vertebrate ERRalpha/beta/gamma. Maternal mRNA of Hr-ERR was detected throughout the entire egg cytoplasm and early embryos. Zygotic Hr-ERR was predominantly expressed in the heart, and at lower levels in muscle, stomach, gonad and digestive glands. Electrophoretic mobility shift assay demonstrated that Hr-ERR directly binds to the estrogen-response element (ERE) and ERR-response element (ERRE). Gene reporter assays also showed that Hr-ERR activates transcription through ERE and ERRE. Hr-ERR-mediated transactivation was modulated by various cofactors for mammalian ERRs, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha and small heterodimer partner. In addition, the ERR antagonists 4-hydroxytamoxifen and diethylstilbestrol inhibited the Hr-ERR-mediated transactivation, whereas Hr-ERR activity on ERE was further induced by genistein, an ERRalpha agonist. Taken together, our results show that Hr-ERR is an unduplicated ERR that however, possesses functional properties common to ERRalpha and not to ERRbeta/gamma.

The orphan receptor ERRalpha interferes with steroid signaling.

The estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily that has been shown to interfere with the estrogen-signaling pathway. In this report, we demonstrate that ERRalpha also cross-talks with signaling driven by other steroid hormones. Treatment of human prostatic cells with a specific ERRalpha inverse agonist reduces the expression of several androgen-responsive genes, in a manner that does not involve perturbation of androgen receptor expression or activity. Furthermore, ERRalpha activates the expression of androgen response elements (ARE)-containing promoters, such as that of the prostate cancer marker PSA, in an ARE-dependent manner. In addition, promoters containing a steroid response element can be activated by all members of the ERR orphan receptor subfamily, and this, even in the presence of antisteroid compounds.

GPER as a Receptor for Endocrine-Disrupting Chemicals (EDCs).

Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that interfere with endogenous hormonal systems at various levels, resulting in adverse health effects. EDCs belong to diverse chemical families and can accumulate in the environment, diet and body fluids, with different levels of persistence. Their action can be mediated by several receptors, including members of the nuclear receptor family, such as estrogen and androgen receptors. The G protein-coupled estrogen receptor (GPER), a seven-transmembrane domain receptor, has also attracted attention as a potential target of EDCs. This review summarizes our current knowledge concerning GPER as a mediator of EDCs' effects.

A Cell-Based Method to Detect Agonist and Antagonist Activities of Endocrine-Disrupting Chemicals on GPER.

Endocrine-disrupting chemicals (EDCs) are exogenous compounds that impact endogenous hormonal systems, resulting in adverse health effects. These chemicals can exert their actions by interfering with several pathways. Simple biological systems to determine whether EDCs act positively or negatively on a given receptor are often lacking. Here we describe a low-to-middle throughput method to screen the agonist/antagonist potential of EDCs specifically on the GPER membrane estrogen receptor. Application of this assay to 23 candidate EDCs from different chemical families reveals the existence of six agonists and six antagonists.

LSD1-ERRα complex requires NRF1 to positively regulate transcription and cell invasion.

Lysine-specific demethylase 1 (LSD1) exerts dual effects on histone H3, promoting transcriptional repression via Lys4 (H3K4) demethylation or transcriptional activation through Lys9 (H3K9) demethylation. These activities are often exerted at transcriptional start sites (TSSs) and depend on the type of enhancer-bound transcription factor (TFs) with which LSD1 interacts. In particular, the Estrogen-Receptor Related α (ERRα) TF interacts with LSD1 and switches its activities toward H3K9 demethylation, resulting in transcriptional activation of a set of common target genes. However, how are the LSD1-TF and, in particular LSD1-ERRα, complexes determined to act at TSSs is not understood. Here we show that promoter-bound nuclear respiratory factor 1 (NRF1), but not ERRα, is essential to LSD1 recruitment at the TSSs of positive LSD1-ERRα targets. In contrast to ERRα, NRF1 does not impact on the nature of LSD1 enzymatic activity. We propose a three factor model, in which the LSD1 histone modifier requires a TSS tethering factor (NRF1) as well as an activity inducer (ERRα) to transcriptionally activate common targets. The relevance of this common network is illustrated by functional data, showing that all three factors are required for cell invasion in an MMP1 (Matrix MetalloProtease 1)-dependent manner, the expression of which is regulated by NRF1/LSD1/ERRα-mediated H3K9me2 demethylation.

Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function.

Thyroid hormone receptor ß1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and ß-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.

Distinct repressive properties of the mammalian and fish orphan nuclear receptors SHP and DAX-1.

It has been suggested that the structure and function of nuclear receptors are evolutionally conserved. Here, we compare the molecular functions of the nile tilapia (Oreochromis niloticus) small heterodimer partner (nSHP/NR0B2) and the Dosage-sensitive sex reversal AHC critical region on X chromosome gene 1 (nDAX-1/NR0B1) with those of human SHP and DAX-1 (hSHP and hDAX-1, respectively). We found that, upon transient cotransfection of human cells, nDAX-1 repressed the activity of tilapia SF-1 (nSF-1) but not that of human SF-1, although the physical interaction with human SF-1 was retained. Similarly, nSHP repressed the activity of nSF-1, whereas hSHP did not, pointing to divergent evolution of SHP/SF-1 in fish and human. We thus propose that the repressive functions of SHP and DAX-1 have been conserved in fish and mammals although with different transcriptional targets and mechanisms. These differences provide new insights into the physiological diversification of atypical orphan nuclear receptors during vertebrate evolution.