Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.     

Kinase suppressor of ras 1 (KSR1) regulates PGC1α and estrogen-related receptor α to promote oncogenic Ras-dependent anchorage-independent growth

Kinase suppressor of ras 1 (KSR1) is a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade that enhances oncogenic Ras signaling. Here we show KSR1-dependent, but ERK-independent, regulation of metabolic capacity is mediated through the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and estrogen-related receptor α (ERRα). This KSR1-regulated pathway is essential for the transformation of cells by oncogenic Ras. In mouse embryo fibroblasts (MEFs) expressing H-Ras(V12), ectopic PGC1α was sufficient to rescue ERRα expression, metabolic capacity, and anchorage-independent growth in the absence of KSR1. The ability of PGC1α to promote anchorage-independent growth required interaction with ERRα, and treatment with an inhibitor of ERRα impeded anchorage-independent growth. In contrast to PGC1α, the expression of constitutively active ERRα (CA-ERRα) was sufficient to enhance metabolic capacity but not anchorage-independent growth in the absence of KSR1. These data reveal KSR1-dependent control of PGC1α- and ERRα-dependent pathways that are necessary and sufficient for signaling by oncogenic H-Ras(V12) to regulate metabolism and anchorage-independent growth, providing novel targets for therapeutic intervention.     

Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.     

ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-α (ERRα) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRα in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRα deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRα deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRα in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRα deficient MSCs and enhanced upon ERRα overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRα. Under adipogenic conditions, ERRα deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRα in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRα may play different roles in bone under different physiological conditions.     

Daidzein promotes the expression of oxidative phosphorylation- and fatty acid oxidation-related genes via an estrogen-related receptor α pathway to decrease lipid accumulation in muscle cells

Estrogen-related receptor (ERR)α regulates genes involved in fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) in muscle. The soy isoflavone daidzein was reported to be a putative ERRα activator, but little is known about its effects on gene expression and FA metabolism. This study aimed to clarify whether daidzein affects FAO- and OXPHOS-related genes thereby modulating intracellular FA metabolism in muscle cells. For this purpose, we used the C2C12 murine muscle cell line. ERRα-expressing C2C12 myotubes were treated with 50 μM daidzein, and gene expression was examined. The expression of FAO genes such as pyruvate dehydrogenase kinase 4 (Pdk4) and acyl-coenzyme A dehydrogenase (Acadm) and that of OXPHOS genes such as ATP synthase F1 subunit beta (Atp5b) and cytochrome c (Cycs) was significantly increased by daidzein, and these effects were partially blocked by an ERRα inhibitor. Using a reporter assay, we showed that daidzein enhanced the promoter activity of these genes and that ERRα responsive elements in the promoter region were necessary for the action of daidzein. Finally, daidzein significantly decreased lipid accumulation in C2C12 myotubes associated with increased oxygen consumption. In conclusion, daidzein decreases lipid deposition in muscle cells by regulating the expression of genes related to FAO and OXPHOS via an ERRα-associated pathway at least in part. These results suggest that daidzein would be a beneficial tool to protect against various diseases caused by muscle lipotoxicity.     

猪雌激素受体关联受体a基因的克隆、表达及其对脂肪细胞脂质聚集的影响

雌激素受体关联受体α(Estrogen-related receptor α,ERRα)是调控机体能量代谢的关键转录调控因子,其在白色脂肪组织中的作用尚不清楚。本研究旨在通过touch down-PCR方法克隆猪ERRα基因的ORF序列;通过Western blotting和细胞免疫荧光染色法分析其在猪各组织及成熟脂肪细胞中的表达模式;利用ERRα特异性抑制剂XCT790处理原代培养的猪成熟脂肪细胞,探讨其对成熟脂肪细胞甘油三酯聚集的影响。结果显示,所克隆的猪ERRα基因ORF序列长1269bp(GenBank Accession No.FJ446485,尚未公布),编码422个氨基酸,其核苷酸和氨基酸序列与其他物种高度同源;ERRα蛋白高表达于猪白色脂肪组织(White adipose tissue,WAT)、肾脏以及心脏中,在脾脏中表达量较低;细胞免疫荧光化学染色显示,ERRα蛋白广泛分布于脂肪细胞的细胞核和胞浆中;XCT790在10μmol/L浓度时显著抑制了ERRα蛋白的表达和成熟脂肪细胞中甘油三酯的聚集。本研究将为有效调控体脂沉积提供新的靶点和理论参考。     

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.     

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P < 0.0125) and creatine uptake (1.8 and 1.6-fold, P < 0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P < 0.05), resulting in a 2.2-fold (P < 0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.