ERRα Activity Research Articles

Loss of estrogen-related receptor α promotes hepatocarcinogenesis development via metabolic and inflammatory disturbances

Estrogen-related receptor α (ERRα) is a key regulator of mitochondrial function and metabolism essential for energy-driven cellular processes in both normal and cancer cells. ERRα has also been shown to mediate bone-derived macrophage activation by proinflammatory cytokines. However, the role of ERRα in cancer in which inflammation acts as a tumor promoter has yet to be investigated. Herein we show that global loss of ERRα accelerates the development of diethylnitrosamine (DEN)-induced hepatocellular carcinoma. Biochemical and metabolomics studies revealed that loss of ERRα promotes hepatocyte necrosis over apoptosis in response to DEN due to a deficiency in energy production. We further show that increased hepatocyte death and associated compensatory proliferation observed in DEN-injured ERRα-null livers is concomitant with increased nuclear factor κB (NF-κB)-dependent transcriptional control of cytokine expression in Kupffer cells. In particular, we demonstrate that loss of ERRα-dependent regulation of the NF-κB inhibitor IκBα leads to enhanced NF-κB activity and cytokine gene activation. Our work thus shows that global loss of ERRα activity promotes hepatocellular carcinoma by independent but synergistic mechanisms in hepatocytes and Kupffer cells, implying that pharmacological manipulation of ERRα activity may have a significant clinical impact on carcinogen-induced cancers.

Estrogen‐Related Receptor ERRα Regulation of Human Hydroxysteroid Sulfotransferase (SULT2A1) Gene Expression in Human Caco‐2 Cells

Human hydroxysteroid sulfotransferase, SULT2A1, is important for xenobiotic detoxification and the maintenance of hydroxysteroid homeostasis. Our published report suggested that estrogen-related receptor ERRα downregulates SULT2A1 in Hep G2 cells. The results shown in this study suggest that ERRα upregulates SULT2A1 transcription in Caco-2 cells. The deletion analysis suggested that SULT2A1 promoter region between -65 and -44 is important for this upregulation. Our further investigation suggested that ERRα binding element, ERRE51, mediates ERRα activation of SULT2A1 promoter transcription in Caco-2 cells. The interaction of ERRE51 with ERRα was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Results also suggest that the difference of constitutive androstane receptor transcription levels in Hep G2 and Caco-2 cells at least partially contribute to the cell type dependent ERRα modulation of SULT2A1 promoter transcription. ERRα regulates human SULT2A1 transcription by competing with other nuclear receptors binding to the DNA-promoter region.

Control of the Human Osteopontin Promoter by ERRα in Colorectal Cancer

Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor α (ERRα) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRα regulates OPN expression in bone prompted us to investigate whether ERRα controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRα than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRα led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRα activity, and expression and mutation of potential ERRα response elements in the proximal promoter of human OPN showed that ERRα and its obligate co-activator, peroxisome proliferator-activated receptor γ co-activator-1 α, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRα in HT29 cells, suggesting that OPN is a direct target of ERRα in colorectal cancer. These findings suggest an additional mechanism by which ERRα participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRα with antagonists as anticancer agents.

Molecular and Genetic Crosstalks between mTOR and ERRα Are Key Determinants of Rapamycin-Induced Nonalcoholic Fatty Liver

mTOR and ERRα are key regulators of common metabolic processes, including lipid homeostasis. However, it is currently unknown whether these factors cooperate in the control of metabolism. ChIP-sequencing analyses of mouse liver reveal that mTOR occupies regulatory regions of genes on a genome-wide scale including enrichment at genes shared with ERRα that are involved in the TCA cycle and lipid biosynthesis. Genetic ablation of ERRα and rapamycin treatment, alone or in combination, alter the expression of these genes and induce the accumulation of TCA metabolites. As a consequence, both genetic and pharmacological inhibition of ERRα activity exacerbates hepatic hyperlipidemia observed in rapamycin-treated mice. We further show that mTOR regulates ERRα activity through ubiquitin-mediated degradation via transcriptional control of the ubiquitin-proteasome pathway. Our work expands the role of mTOR action in metabolism and highlights the existence of a potent mTOR/ERRα regulatory axis with significant clinical impact.

Molecular Pathways: The Metabolic Regulator Estrogen-Related Receptor α as a Therapeutic Target in Cancer

The estrogen-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily of transcription factors whose activity is regulated by the expression level and/or activity of its obligate coregulators, peroxisome proliferator-activated receptor γ coactivator-1 α and β (PGC-1α or PGC-1β). Under normal physiologic conditions, and in responding to different environmental stimuli, the ERRα/PGC-1 complex is involved in regulating metabolic homeostasis under conditions of high energy demand in brown adipocytes, proliferating T cells, and muscle. Interestingly, increased expression and activity of the ERRα/PGC-1 axis has also been shown to correlate with unfavorable clinical outcomes in both breast and ovarian tumors. The observation that ERRα activity is manifest in all breast tumor subtypes with particularly high activity being evident in ERα-negative, HER2-positive, and triple-negative breast cancers has raised significant interest in targeting this receptor for the treatment of those breast cancers for which therapeutic options are limited.

Abstract 292: Isoform-Specific Expression of Estrogen-Related Receptors Regulates Cardiomyocyte Metabolic Adaptation to Oxidative Stress

In the ischemic heart, metabolic adaptation of cardiomyocytes exposed to prolonged hypoxia is critical to sustain myocardial energy production. Metabolic gene expression is regulated by Estrogen-Related Receptor (ERR) transcription factors and Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) co-activators. ERRs do not require ligand to achieve an active conformation and regulate an array of target genes involved in myocardial energy metabolism. These studies aim to determine the regulatory mechanisms governing expression of ERR isoforms (ERRα and ERRβ) in cardiomyocytes undergoing oxidative stress. We hypothesize that metabolic energy production in myocardium initially adapts to hypoxia by inducing transient ERRβ expression, and maintains this compensatory expression through induction of sustained ERRα expression. Adult feline cardiomyocytes in primary culture were electrically stimulated to contract continuously at 1 Hz under normoxia (21% [O2]), hypoxia (0.5% [O2]), or hypoxia followed by reoxygenation. Expression of ERR, PGC-1 and target genes was measured by QRT-PCR (n≥5). QRT-PCR arrays were used to screen for prospective target genes. In response to 24 hours of hypoxia, mRNA levels increased for ERRα (3-fold by 24 hours), ERRβ (11-fold by 12 hours), PGC-1α (16-fold by 24 hours) and PGC-1β (8-fold by 24 hours). Subsequent reoxygenation for 24 hours decreased ERRβ, PGC-1α and PGC-1β mRNA levels to that of normoxia controls. However, reoxygenation did not markedly decrease (∼25%; 0.7 of 3.0-fold) ERRα mRNA levels. Cytochrome C Oxidase Subunit VIc (Cox6c) and Fatty Acid Binding Protein 3 (Fabp3) were identified as potential ERRα target genes in adult cardiomyocytes based on correlation of expression. Moreover, hypoxia-induced increases in Cox6c and Fabp3 mRNA levels were partially blocked by treatment with XCT-790, an inhibitor of ERRα activity. We conclude that hypoxia and subsequent reoxygenation elicit isoform-specific changes in ERR and PGC-1 mRNA expression in adult cardiomyocytes. This observed regulation of expression for ERR isoforms and their target genes may play a critical role in metabolic adaptation to oxidative stress.

Abstract 1305: Alternative splicing of the metabolic regulator Estrogen-Related Receptor alpha (ERRα) in colon carcinogenesis

Abstract The nuclear receptor ERRα acts principally in pair with its coactivator PGC-1α as a regulator of metabolic processes particularly in tissues subjected to high-energy demand. Besides its implication in energy metabolism and mitochondrial biogenesis, ERRα was recently associated with tumorigenesis. Increased expression of ERRα was noted in different cancerous tissues as breast, ovary and colon. Also, a new form of ERRα lacking the exon5 (ERRα Δ5) was recently identified and suggested to be a dominant negative of ERRα and interestingly to be differently expressed in normal and cancerous tissues. Since a protumorigenic role is emerging for this metabolic regulator, we investigated the role of ERRα and its spliced form ERRα β5 in colon carcinogenesis. METHODS: shRNA-mediated silencing of ERRα and overexpression of ERRα β5 were performed by lentivirus infection in DLD1 and HCT116 human colon cancer cells. The interaction between both proteins was investigated as well as their respective roles in various tumorigenic processes. RESULTS: Silencing of ERRα by shRNA reduced proliferation of DLD1 and HCT116 cells as measured by growth kinetics and growth assays in soft agar and reduced tumor formation in NUDE mice. Furthermore, FACS-scan analysis revealed that ERRα silencing delays G1-S phase transition in colon cancer cells. Differential expression of a splice variant of ERRα (ERRα Δ5) in cancer vs normal tissues would support the importance of ERRα in colorectal carcinogenesis. This is the case at least for colon tumors as cancerous samples display lower amount of ERRα β5 and higher amount of ERRα compared to adjacent normal tissues. Also, the ERRα β5 expression is surprisingly regulated by proteosomal degradation in normal and colon cancer cell lines and this could explain the low abundance of the ERRα spliced form. Interaction between both variants has been characterized by immunoprecipitation assays, confirming the conserved dimerization potential of ERRα β5 variant with ERRα. Furthermore, luciferase assays revealed that ERRα β5 is transcriptionaly inactive and its expression inhibits the transcriptional activity of ERRα on its target genes, suggesting that this variant acts as a dominant negative for ERRα. Interestingly, the reexpression of ERRα β5 in colon cancer cells reduces soft agar colony formation. CONCLUSION: Strong ERRα expression is associated with cancerous tissues and is required for the intense proliferation of colon cancer cells. Interestingly, the poorly active splice variant ERRα β5 is lost in cancerous colon tissues and has antiproliferative properties when reintroduced in colon cancer cells. Since this splice variant could interact with ERRα and inhibit its activity, ERRα β5 reduction of expression in cancerous tissue could allow ERRα to fully promote colon cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1305. doi:1538-7445.AM2012-1305

Negative regulation of ERRα by a novel nucleolar protein

The regulation of estrogen-related receptor (ERR) transcriptional activity is poorly understood. To explore the underlying mechanism, we sought to isolate ERRα-binding protein(s). In a yeast two-hybrid screen, we identified a novel protein that has been characterized as a retinoic acid resistance factor (RaRF) (manuscript in-preparation). A specific interaction between RaRF and ERRα was confirmed in a GST pull-down assay in vitro and immunoprecipitation (IP) in mammalian cells. Further yeast two-hybrid assays and IP analyses indicated that the C-terminus of ERRα is required for RaRF binding. Consistent with our interaction data, transfection of RaRF significantly reduced the ability of ERRα, but not ERRγ, to transactivate an ERR-responsive luciferase reporter. In contrast, down-regulation of RaRF using shRNA increased ERRα activity without affecting that of ERRγ. RaRF was subsequently shown to repress the expression of the ERR target gene pS2. Further fluorescence microscopy revealed that ERRα or ERRγ is normally expressed in the nucleoplasm, with ERRα, but not ERRγ, translocating to the nucleolus when RaRF is expressed. Taken together, our data suggest that RaRF sequesters ERRα in the nucleolus through a specific interaction, thereby inhibiting its transcriptional activity.

The Metabolic Regulator ERRα, a Downstream Target of HER2/IGF-1R, as a Therapeutic Target in Breast Cancer

A genomic signature designed to assess the activity of the estrogen-related receptor alpha (ERRα) was used toprofile more than 800 breast tumors, revealing a shorter disease-free survival in patients with tumors exhibiting elevated receptor activity. Importantly, this signature also predicted the ability of an ERRα antagonist, XCT790, to inhibit proliferation in cellular models of breast cancer. Using a chemical genomic approach, it was determined that activation of the Her2/IGF-1R signaling pathways and subsequent C-MYC stabilization upregulate the expression of peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1β), an obligate cofactor for ERRα activity. PGC-1β knockdown in breast cancer cells impaired ERRα signaling and reduced cell proliferation, implicating a functional role for PGC-1β/ERRα in the pathogenesis of breast cancers.

Genomic Convergence among ERRα, PROX1, and BMAL1 in the Control of Metabolic Clock Outputs

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes.

Protein kinase C epsilon affects mitochondrial function through estrogen-related receptor alpha

Members of the protein kinase C (PKC) family have been implicated in controlling cell proliferation, differentiation, and motility. Many of these processes are energy demanding. How PKCs affect mitochondrial function to regulate energy production is not well defined. Using an inhibitor Gö6983 with broad specificity, we found that inhibiting PKCs reduced mitochondrial mass and altered mitochondrial function characterized by elevations in mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) levels. These alterations indicated that Gö6983 suppressed the activities of mitochondrial regulators such as estrogen-related receptor α (ERRα). Indeed, Gö6983 dose-dependently suppressed the expression levels of ERRα-target genes peroxisome proliferator-activated receptor α (PPARα) and medium-chain acyl-CoA dehydrogenase (MCAD). Conversely, PKC activator phorbol ester (PMA) enhanced the expression level of another ERRα-target gene pyruvate dehydrogenase kinase 4 (PDK4). This PMA-mediated induction of PDK4 was blunted by an ERRα inverse agonist XCT-790, suggesting that ERRα plays a role in mediating the effects of PKCs on mitochondrial function. By over-expressing constitutively active forms of PKCs, we found that PKCε preferentially stimulated the transcription activity of ERRα. Through mutating residues on ERRα, we established that this PKCε-induced ERRα activity involves threonine 106, serine 110, and threonine 124 of ERRα. Collectively, these pieces of evidence suggest that ERRα plays an important role down-stream of PKCε to regulate mitochondrial homeostasis.

Abstract 2045: Intriguing role of estrogen-related receptor alpha (ERRα) in colorectal cancer

Abstract Besides its well-known function in energetic metabolism, the nuclear receptor Estrogen-Related Receptor Alpha (ERRα) has also been shown to be overexpressed in breast, ovarian, uterine, prostate and colon cancer and to be associated to a poor prognosis in some of these cancer types. Interestingly, an ERRα splice variant mainly expressed in non cancerous tissues has been reported. This variant missing the fifth exon (ERRα Δ5) is still expected to interact with DNA and form dimers but cannot recruit coactivators, therefore being devoid of any transcriptional activity. AIM: Determine the role of ERRα as well as ERRα Δ5 in intestinal tumorigenesis. METHODS: shRNA-mediated silencing of ERRα and overexpression of ERRα Δ5 were performed by lentivirus infection in DLD1 and HCT116 human colon cancer cells. The interaction between both proteins was investigated as well as their respective roles in various tumorigenic processes. RESULTS: Silencing of ERRα by shRNA reduced proliferation of DLD1 and HCT116 cells as measured by growth kinetics and anchorage-independent growth assays in soft agar. We also observed that ERRα knockdown cells grew into smaller tumors when subcutaneously implanted in NUDE mice. Furthermore, FACS-scan analysis revealed that ERRα silencing delays G1 to S phase transition in colon cancer cells. Differential expression of a splice variant of ERRα (ERRα Δ5) in cancer vs normal tissues would support the importance of ERRα role in colorectal carcinogenesis. This is the case at least for colon tumors as cancerous samples display lower amount of ERRα Δ5 and higher amount of ERRα compared to adjacent paired normal tissues, as measured by Western Blot analysis. Interaction between both variants has been characterized by immunoprecipitation assays, confirming the conserved dimerization potential of ERRα Δ5 variant with the full length ERRα. Furthermore, luciferase assays revealed that ERRα Δ5 is transcriptionaly inactive and its expression inhibits the transcriptional activity of ERRα on its target genes, suggesting that this variant acts as a dominant negative for ERRα. Interestingly, the reintroduction of ERRα Δ5 in human colon cancer cells leads to the reduction of cell growth and soft agar colony formation. CONCLUSION: Strong ERRα expression is associated with cancerous tissues and is required for the intense proliferation of colon cancer cells. Interestingly, the poorly active splice variant ERRα Δ5 is lost in cancerous colon tissues and has antiproliferative properties when reintroduced in colon cancer cells. Since this splice variant could interact with ERRα and inhibit its activity, ERRα Δ5 reduction of expression in cancerous tissue could allow ERRα to fully promote colon cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2011-2045

Estrogen-Related Receptor α Expression and Function Are Associated With Vascular Endothelial Growth Factor in Human Cervical Cancer

Estrogen-related receptor α (ERRα), one of orphan nuclear receptors with an unknown ligand, is expressed in various types of cancer. Increased ERRα levels are associated with a higher risk of recurrence and poor clinical outcome in breast cancer, suggesting that ERRα could be a negative prognostic factor. Recently, it has been suggested that vascular endothelial growth factor (VEGF) could be one of the transcriptional targets of ERRα in breast cancer. Here, we examined the expression of ERRα and the association of ERRα with VEGF in uterine cervical cancer cells and tissues. We evaluated the expression of ERRα and VEGF by immunohistologic analysis using specimens from 40 patients with invasive cervical cancer. We also evaluated the VEGF promoter activity of ERRα in cervical cancer cell lines by transfection and luciferase assay. We overexpressed or knocked down ERRα and examined VEGF expression by real-time polymerase chain reaction. Finally, cell proliferation assay was performed to examine whether ERRα affects tumor growth in cervical cancer. Immunohistologic analysis demonstrated that ERRα expression in cervical cancer tissues was higher than that in noncancerous tissues and that there was a positive association between ERRα and VEGF expression in cancer tissues (P < 0.05). We showed that ERRα stimulated the VEGF promoter activity in cervical cancer cell lines. We further showed the overexpression and knockdown of ERRα-regulated VEGF expression level by real-time polymerase chain reaction. Moreover, we showed that ERRα and VEGF knockdown by small interfering RNA or an inverse agonist of ERRα, XCT 790, could suppress cell growth compared with control cells in cervical cancer. We have provided compelling evidence that ERRα affects VEGF expression and tumor growth in cervical cancer. These results justify further investigation into the use of ERRα as a therapeutic target for patients with uterine cervical cancer.

Extracellular Signal-regulated Kinase 8 (ERK8) Controls Estrogen-related Receptor α (ERRα) Cellular Localization and Inhibits Its Transcriptional Activity

ERK8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERRα as a novel ERK8-interacting protein. As a consequence of such interaction, ERK8 induces CRM1-dependent translocation of ERRα to the cytoplasm and inhibits its transcriptional activity. Also, we identify in ERK8 two LXXLL motifs, typical of agonist-bound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERRα and to regulate its cellular localization and transcriptional activity. Ultimately, we demonstrate that ERK8 is able to counteract, in immortalized human mammary cells, ERRα activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for ERK8 as a bona fide ERRα corepressor, involved in control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the regulation of the biological activities of this nuclear receptor.

WNT11 Expression Is Induced by Estrogen-Related Receptor α and β-Catenin and Acts in an Autocrine Manner to Increase Cancer Cell Migration

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, β-catenin (β-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by β-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or β-cat. Furthermore, silencing of ERRα and β-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/β-cat-dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/β-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and β-cat that influences the migratory capacity of cancer cells.

Involvement of the orphan nuclear estrogen receptor-related receptor α in osteoclast adhesion and transmigration

The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte–macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.

An Acetylation Switch Modulates the Transcriptional Activity of Estrogen-Related Receptor α

Posttranslational modifications are instrumental to achieve gene- and tissue-specific regulatory outcomes by transcription factors. Nuclear receptors are dynamically modulated by several types of posttranslational modifications including phosphorylation, methylation, acetylation, ubiquitination, and sumoylation. The estrogen-related receptor alpha (ERRalpha, NR3B1) is phosphorylated on multiple sites, and sumoylated in the amino-terminal region in a phosphorylation-dependent manner. Here we demonstrate that ERRalpha interacts with and is acetylated by p300 coactivator associated factor (PCAF) in vitro and in mouse liver. Purified PCAF acetylated the DNA-binding domain of ERRalpha on four highly-conserved lysines. In addition, coexpression of PCAF reduced the transcriptional activity of ERRalpha and, reciprocally, a deacetylase screen identified histone deacetylase 8 (HDAC8) and sirtuin 1 homolog (Sirt1) as independent enhancers of ERRalpha transcriptional function. HDAC8 and Sirt1 were also demonstrated to interact directly with ERRalpha in vivo and to deacetylate and increase the DNA binding affinity of ERRalpha in vitro. The removal of PCAF increases the DNA binding of ERRalpha in vivo, whereas the removal of Sirt1 and HDAC8 decreases it as assessed by chromatin immunoprecipitation assay. Altogether, our results show that ERRalpha is an acetylated protein and imply the existence of a dynamic acetylation/deacetylation switch involved in the control of ERRalpha transcriptional activity.

Estrogen Receptor-Related Receptor α Mediates Up-Regulation of Aromatase Expression by Prostaglandin E2 in Prostate Stromal Cells

Estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRalpha is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRalpha, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRalpha, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRalpha expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRalpha activity by chlordane (an antagonist of ERRalpha) or small interfering RNA knockdown of ERRalpha blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRalpha significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRalpha directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRalpha and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17Beta-estradiol concentration in WPMY-1 medium was up-regulated by ERRalpha expression, and that was further increased by PGE2. Our results provided evidence that ERRalpha contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRalpha in estrogen-related prostatic diseases.

Human fetal mesenchymal stem cells differentiate into brown and white adipocytes: a role for ERRα in human UCP1 expression

We have investigated the ability of fetal mesenchymal stem cells (fMSCs) to differentiate into brown and white adipocytes and compared the expression of a number of marker genes and key regulatory factors. We have shown that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and murine adipocyte models, including induction of PPARγ2 and FABP4. Notably we found that the pre-adipocyte marker Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire pre-adipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adipocytes and others into white. Detailed investigation of one isolate showed that the novel brown fat determining factor PRDM16 is expressed both before and after differentiation. Importantly these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRα, highlighting a novel role for ERRα in human brown fat. Thus fetal MSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable insights into the characteristics of human brown fat.

Physiological Genomics Identifies Estrogen-Related Receptor α as a Regulator of Renal Sodium and Potassium Homeostasis and the Renin-Angiotensin Pathway

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor highly expressed in the kidney, an organ playing a central role in blood pressure regulation through electrolyte homeostasis and the renin-angiotensin system. Physiological analysis revealed that, relative to wild-type mice, ERRα null mice are hypotensive despite significant hypernatremia, hypokalemia, and slight hyperreninemia. Using a combination of genome-wide location analysis and expression profiling, we demonstrate that ERRα regulates the expression of channels involved in renal Na+ and K+ handling (Scnn1a, Atp1a1, Atp1b1) and altered in Bartter syndrome (Bsnd, Kcnq1). In addition, ERRα regulates the expression of receptors implicated in the systemic regulation of blood pressure (Ghr, Gcgr, Lepr, Npy1r) and of genes within the renin-angiotensin pathway (Ren1, Agt, Ace2). Our study thus identifies ERRα as a pleiotropic regulator of renal control of blood pressure, renal Na+/K+ homeostasis, and renin-angiotensin pathway and suggests that modulation of ERRα activity could represent a potential avenue for the management of hypertension.