Treatment with retinoic acid (RA) is known to produce complex teratogenic effects in vertebrates, and its presence in the developing embryo as an endogenous substance has led to the suggestion that RA might be a natural morphogenetic agent. Although our understanding of the molecular mechanism of RA action has improved considerably with the identification of nuclear receptors for RA (RARs) and RA-responsive genes, the exact relationship between the proposed morphogenetic activity of RA and its teratogenic effects remains to be characterized. Here, we show that a RA response element (RARE) present in the RARI3 gene can direct specific spatial and temporal expression of an hsplacZ transgene during mouse embryogenesis. In the early embryo, the transgene is expressed in a specific anterior-posterior domain that is completely obliterated by treatment of pregnant mice with teratogenic doses of RA. The expression of the transgene becomes more restricted as organogenesis progresses and mimics closely the reported expression of the RARI$ gene. These results suggest that, in vivo, some of the morphogenetic effects of RA could be mediated through localized transcriptional activity controlled by the various RARs. The specific pattern of expression of the RAREhsplacZ transgene does not correlate with the proposed sites of action of RA as defined by its teratogenic effects but does support a role for RA in early anterior-posterior patterning along the body axis.

Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBalpha and RORalpha, play important roles in the transcription of the clock gene Bmal1. The authors now considerably extend these findings with the demonstration that all members of the REV-ERB (alpha and beta) and ROR (alpha, beta, and gamma) families repress and activate Bmal1 transcription, respectively. The authors further show that transcription of Bmal1 is the result of competition between REV-ERBs and RORs at their specific response elements (RORE). Moreover, they demonstrate that Reverb genes are similarly expressed in the thymus, skeletal muscle, and kidney, whereas Ror genes present distinct expression patterns. Thus, the results indicate that all members of the REV-ERB and ROR families are crucial components of the molecular circadian clock. Furthermore, their strikingly different patterns of expression in nervous and peripheral tissues provide important insights into functional differences between circadian clocks within the organism.

Transcriptional control of cellular energy metabolic pathways is achieved by the coordinated action of numerous transcription factors and associated coregulators. Several members of the nuclear receptor superfamily have been shown to play important roles in this process because they can translate hormonal, nutrient, and metabolite signals into specific gene expression networks to satisfy energy demands in response to distinct physiological cues. Estrogen-related receptor (ERR) alpha, ERRbeta, and ERRgamma are nuclear receptors that have yet to be associated with a natural ligand and are thus considered as orphan receptors. However, the transcriptional activity of the ERRs is exquisitely sensitive to the presence of coregulatory proteins known to be essential for the control of energy homeostasis, and for all intents and purposes, these coregulators function as protein ligands for the ERRs. In particular, functional genomics and biochemical studies have shown that ERRalpha and ERRgamma operate as the primary conduits for the activity of members of the family of PGC-1 coactivators. As transcription factors, the ERRs control vast gene networks involved in all aspects of energy homeostasis, including fat and glucose metabolism as well as mitochondrial biogenesis and function. Phenotypic analyses of knockout mouse models have shown that all three ERRs are indispensable for proper development and/or survival of the organism when subjected to a variety of physiological challenges. The focus of this review is on the recent and rapid advances in understanding the functions of the ERRs in regulating bioenergetic pathways, with an emphasis on their roles in the specification of energetic properties required for cell- and tissue-specific functions.

Orphan nuclear receptor ERRalpha (NR3B1) is recognized as a key regulator of mitochondrial biogenesis, but it is not known whether ERRalpha and other ERR isoforms play a broader role in cardiac energetics and function. We used genome-wide location analysis and expression profiling to appraise the role of ERRalpha and gamma (NR3B3) in the adult heart. Our data indicate that the two receptors, acting as nonobligatory heterodimers, target a common set of promoters involved in the uptake of energy substrates, production and transport of ATP across the mitochondrial membranes, and intracellular fuel sensing, as well as Ca(2+) handling and contractile work. Motif-finding algorithms assisted by functional studies indicated that ERR target promoters are enriched for NRF-1, CREB, and STAT3 binding sites. Our study thus reveals that the ERRs orchestrate a comprehensive cardiac transcriptional program and further suggests that modulation of ERR activities could be used to manage cardiomyopathies.

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Estrogen-related receptors (ERRs) are orphan nuclear receptors activated by the transcriptional coactivator peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator 1␣ (PGC-1␣), a critical regulator of cellular energy metabolism. However, metabolic target genes downstream of ERR␣ have not been well defined. To identify ERR␣-regulated pathways in tissues with high energy demand such as the heart, gene expression profiling was performed with primary neonatal cardiac myocytes overexpressing ERR␣. ERR␣ upregulated a subset of PGC-1␣ target genes involved in multiple energy production pathways, including cellular fatty acid transport, mitochondrial and peroxisomal fatty acid oxidation, and mitochondrial respiration. These results were validated by independent analyses in cardiac myocytes, C 2 C 12 myotubes, and cardiac and skeletal muscle of ERR␣ ؊/؊ mice. Consistent with the gene expression results, ERR␣ increased myocyte lipid accumulation and fatty acid oxidation rates. Many of the genes regulated by ERR␣ are known targets for the nuclear receptor PPAR␣, and therefore, the interaction between these regulatory pathways was explored. ERR␣ activated PPAR␣ gene expression via direct binding of ERR␣ to the PPAR␣ gene promoter. Furthermore, in fibroblasts null for PPAR␣ and ERR␣, the ability of ERR␣ to activate several PPAR␣ targets and to increase cellular fatty acid oxidation rates was abolished. PGC-1␣ was also shown to activate ERR␣ gene expression. We conclude that ERR␣ serves as a critical nodal point in the regulatory circuitry downstream of PGC-1␣ to direct the transcription of genes involved in mitochondrial energy-producing pathways in cardiac and skeletal muscle.The essential role of nuclear receptors in regulating various cellular metabolic pathways is becoming increasingly evident. In recent years, various nuclear receptors that do not respond to classical endocrine ligands, including peroxisome proliferator-activated receptors (PPARs), liver X receptors, farnesoid X receptors, and retinoid X receptors, have been shown to be activated by low-affinity diet-derived ligands (6,11,26,44). Activation of these receptors by metabolite ligands such as fatty acids, oxysterols, and bile acids elicits downstream transcriptional regulation of pathways involved in synthesis and catabolism of these ligands. The remaining receptors, designated orphan receptors because endogenous ligands have not been identified, comprise the largest subcategory of nuclear receptors. It is likely that orphan receptors serve additional roles in regulating intermediary metabolism. Linking orphan receptors to target genes is an important goal in the field of nuclear receptor biology. Target gene profiling will also provide insights for determining what metabolites serve as endogenous ligands for these receptors and, in turn, for developing pharmacologic interventions designed to regulate cellular metabolism.One group of orphan receptors recently identified as candidate regulators of cellular metabolism are the estrogen-related recepto...

The transcriptional coactivator PGC-1␣ is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1␣ expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1␣ in C 2 C 12 myotubes induced PDK4 mRNA and protein expression. PGC-1␣-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1␣-responsive element bound the estrogen-related receptor ␣ (ERR␣), a recently identified component of the PGC-1␣ signaling pathway. In addition, PGC-1␣ was shown to activate ERR␣ expression. Chromatin immunoprecipitation assays confirmed that PGC-1␣ and ERR␣ occupied the mPDK4 promoter in C 2 C 12 myotubes. Additionally, transfection studies using ERR␣-null primary fibroblasts demonstrated that ERR␣ is required for PGC-1␣-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1␣ on PDK4 gene transcription, overexpression of PGC-1␣ in C 2 C 12 myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1␣/ERR␣ target and suggest a mechanism whereby PGC-1␣ exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.

The estrogen-related receptor ␣ (ERR␣) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERR␣ and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERR␣, we generated and analyzed Estrra-null (ERR␣ ؊/؊ ) mutant mice. Here we show that ERR␣ ؊/؊ mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERR␣ might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERR␣ functions as a metabolic regulator and that the ERR␣ ؊/؊ mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.Nuclear receptors are ligand-regulated transcription factors that control key pathways required for normal development and maintenance of homeostasis throughout life (37). Nuclear receptors now comprise a family of 48 genes in mice and humans that encode structurally and functionally related proteins. However, the existence of fewer than 10 receptors had been predicted by classic physiological and biochemical studies (10). Since the discovery of many nuclear receptors had not been anticipated and thus is not linked to recognized natural ligands, these new gene products were referred to as orphan nuclear receptors. During the last decade, extensive study of this gene family revealed that orphan nuclear receptors control essential developmental and metabolic functions in response to natural ligands as diverse as steroid hormones, retinoic acids, leukotrienes, bile acids, cholesterol metabolites, and long-chain fatty acids (reviewed in references 5, 26, and 48). In addition, orphan nuclear receptors have been shown to react to the presence of exogenous ligands such as pesticides (15, 70), phenobarbital (53, 61), and a wide variety of xenobiotic agents and drugs (2,24,39,55,66,68,69). Gene deletion analyses in mice have been particularly useful to uncover biological functions of orphan nuclear receptors. Nuclear orphan receptors have been shown to participate in the development and/or maintenance of the placenta, somitogenesis, brain, heart, hypothalamus-pituitary axis, immune system, and pathways controlling s...