Janice M. Huss

Constitutive activities of estrogen-related receptors: Transcriptional regulation of metabolism by the ERR pathways in health and disease

The estrogen-related receptors (ERRs) comprise a small group of orphan nuclear receptor transcription factors. The ERRα and ERRγ isoforms play a central role in the regulation of metabolic genes and cellular energy metabolism. Although less is known about ERRβ, recent studies have revealed the importance of this isoform in the maintenance of embryonic stem cell pluripotency. Thus, ERRs are essential to many biological processes. The development of several ERR knockout and overexpression models and the application of advanced functional genomics have allowed rapid advancement of our understanding of the physiology regulated by ERR pathways. Moreover, it has enabled us to begin to delineate the distinct programs regulated by ERRα and ERRγ that have overlapping effects on metabolism and growth. The current review primarily focuses on the physiologic roles of ERR isoforms related to their metabolic regulation; therefore, the ERRα and ERRγ are discussed in the greatest detail. We emphasize findings from gain- and loss-of-function models developed to characterize ERR control of skeletal muscle, heart and musculoskeletal physiology. These models have revealed that coordinating metabolic capacity with energy demand is essential for seemingly disparate processes such as muscle differentiation and hypertrophy, innate immune function, thermogenesis, and bone remodeling. Furthermore, the models have revealed that ERRα- and ERRγ-deficiency in mice accelerates progression of pathologic processes and implicates ERRs as etiologic factors in disease. We highlight the human diseases in which ERRs and their downstream metabolic pathways are perturbed, including heart failure and diabetes. While no natural ligand has been identified for any of the ERR isoforms, the potential for using synthetic small molecules to modulate their activity has been demonstrated. Based on our current understanding of their transcriptional mechanisms and physiologic relevance, the ERRs have emerged as potential therapeutic targets for treatment of osteoporosis, muscle atrophy, insulin resistance and heart failure in humans.

Cardiac lipin 1 expression is regulated by the peroxisome proliferator activated receptor γ coactivator 1α/estrogen related receptor axis

Lipin family proteins (lipin 1, 2, and 3) are bifunctional intracellular proteins that regulate metabolism by acting as coregulators of DNA-bound transcription factors and also dephosphorylate phosphatidate to form diacylglycerol [phosphatidate phosphohydrolase activity] in the triglyceride synthesis pathway. Herein, we report that lipin 1 is enriched in heart and that hearts of mice lacking lipin 1 (fld mice) exhibit accumulation of phosphatidate. We also demonstrate that the expression of the gene encoding lipin 1 (Lpin1) is under the control of the estrogen-related receptors (ERRs) and their coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α, ERRα, or ERRγ overexpression increased Lpin1 transcription in cultured ventricular myocytes and the ERRs were associated with response elements in the first intron of the Lpin1 gene. Concomitant RNAi-mediated knockdown of ERRα and ERRγ abrogated the induction of lipin 1 expression by PGC-1α overexpression. Consistent with these data, 3-fold overexpression of PGC-1α in intact myocardium of transgenic mice increased cardiac lipin 1 and ERRα/γ expression. Similarly, injection of the β2-adrenergic agonist clenbuterol induced PGC-1α and lipin 1 expression, and the induction in lipin 1 after clenbuterol occurred in a PGC-1α-dependent manner. In contrast, expression of PGC-1α, ERRα, ERRγ, and lipin 1 was down-regulated in failing heart. Cardiac phosphatidic acid phosphohydrolase activity was also diminished, while cardiac phosphatidate content was increased, in failing heart. Collectively, these data suggest that lipin 1 is the principal lipin protein in the myocardium and is regulated in response to physiologic and pathologic stimuli that impact cardiac metabolism.

Estrogen-related receptor-α (ERRα) deficiency in skeletal muscle impairs regeneration in response to injury

The estrogen‐related receptor‐α (ERRα) regulates mitochondrial biogenesis and glucose and fatty acid oxidation during differentiation in skeletal myocytes. However, whether ERRα controls metabolic remodeling during skeletal muscle regeneration in vivo is unknown. We characterized the time course of skeletal muscle regeneration in wild‐type (M‐ERRα WT) and muscle‐specific ERRα–/– (M‐ERRα–/–) mice after injury by intramuscular cardiotoxin injection. M‐ERRα–/‐ mice exhibited impaired regeneration characterized by smaller myofibers with increased centrally localized nuclei and reduced mitochondrial density and cytochrome oxidase and citrate synthase activities relative to M‐ERRαWT. Transcript levels of mitochondrial transcription factor A, nuclear respiratory factor‐2α, and peroxisome proliferator‐activated receptor (PPAR)‐γ coactivator (PGC)‐1β, were downregulated in the M‐ERRα–/– muscles at the onset of myogenesis. Furthermore, coincident with delayed myofiber recovery, we observed reduced muscle ATP content (–45% vs. M‐ERRαWT) and enhanced AMP‐activated protein kinase (AMPK) activation in M‐ERRα–/– muscle. We subsequently demonstrated that pharmacologic postinjury AMPK activation was sufficient to delay muscle regeneration in WT mice. AMPK activation induced ERRα transcript expression in M‐ERRαWT muscle and in C2C12 myotubes through induction of the Esrra promoter, indicating that ERRα may control gene regulation downstream of the AMPK pathway. Collectively, these results suggest that ERRα deficiency during muscle regeneration impairs recovery of mitochondrial energetic capacity and perturbs AMPK activity, resulting in delayed myofiber repair.—LaBarge, S., McDonald, M., Smith‐Powell, L., Auwerx, J., Huss, J. M. Estrogen‐related receptor‐α (ERRα) deficiency in skeletal muscle impairs regeneration in response to injury. FASEB J. 28, 1082–1097 (2014). www.fasebj.org

A PPARα Promoter Variant Impairs ERR-Dependent Transactivation and Decreases Mortality after Acute Coronary Ischemia in Patients with Diabetes

Activation of peroxisome proliferator-activated receptor alpha (PPARα) occurs in animal models of diabetes (DM) and is implicated in pathological responses to myocardial ischemia. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in the PPARα gene promoter (PPARA −54,642 G>A; rs135561) that altered the consensus sequence for a nuclear receptor binding site. Electrophoretic mobility shift assays showed that the domain bound two known PPARA transcriptional activators, estrogen-related receptor (ERR)-α and -γ and that PPARA G bound with greater affinity than PPARA A (>2-fold; P<0.05). Likewise, promoter-reporter analyses showed enhanced transcriptional activity for PPARA G vs. PPARA A for both ERR-α and -γ (3.1 vs.1.9-fold; P<0.05). Since PPARα activation impairs post-ischemic cardiac function in experimental models of DM, we tested whether decreased PPARA transcription in PPARA A carriers favorably impacted outcome after acute coronary ischemia in 705 patients hospitalized with acute coronary syndromes (ACS; 552 Caucasian, 106 African American). PPARA A allele frequencies were similar to non-diseased subjects. However, PPARA genotype correlated with 5-year mortality in diabetic (22.2% AA vs. 18.8% AG vs. 39.5% GG; P = 0.008), but not non-diabetic (P = 0.96) subjects (genotype by diabetes interaction P = 0.008). In the diabetic ACS subjects, PPARA A carriers had strikingly reduced all-cause mortality compared to PPARA G homozygotes, (unadjusted HR 0.44, 95% CI 0.26–0.75; P = 0.003; adjusted HR 0.48, 95% CI 0.27–0.83; P = 0.009). Consistent with previous descriptions of PPARα in experimental models and human disease, we describe a novel PPARA promoter SNP that decreases transcriptional activation of PPARA and protects against mortality in diabetic patients after ACS.

Annual life-stage regulation of lipid metabolism and storage and association with PPARs in a migrant species: the gray catbird (Dumetella carolinensis).

ABSTRACT The annual cycle of a migrating bird involves metabolically distinct stages of substantial fatty acid storage and periods of increased fatty acid mobilization and utilization, and thus requires a great deal of phenotypic flexibility. Specific mechanisms directing stage transitions of lipid metabolism in migrants are largely unknown. This study characterized the role of the PPARs (peroxisome proliferator-activated receptors) in regulating migratory adiposity of the gray catbird (Dumetella carolinensis). Catbirds increased adipose storage during spring and autumn migration and showed increased rates of basal lipolysis during migration and tropical overwintering. Expression of the PPAR target genes involved in fat uptake and storage, FABPpm and PLIN3, increased during pre-migratory fattening. We found significant correlation between PPARγ and target gene expression in adipose but little evidence that PPARα expression levels drive metabolic regulation in liver during the migratory cycle. Summary: Changes in energetics related to the migratory cycle span organismal, tissue and molecular endpoints, and these seasonal adjustments involve the PPAR family of nuclear receptors and their target genes.

t-Darpp Activates IGF-1R Signaling to Regulate Glucose Metabolism in Trastuzumab-Resistant Breast Cancer Cells

Purpose: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp overexpression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers. Experimental Design: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival. To assess t-Darpps metabolic effects, we used the Seahorse flux analyzer to measure glucose metabolism in trastuzumab-resistant SK-BR-3 cells (SK.HerR) that have high endogenous t-Darpp levels and SK.tDrp cells that stably overexpress exogenous t-Darpp. To investigate t-Darpps mechanism of action, we evaluated t-Darpp:IGF-1R complexes by coimmunoprecipitation and proximity ligation assays. We used pathway-specific inhibitors to study the dependence of t-Darpp effects on IGF-1R signaling. We used siRNA knockdown to determine whether glucose reliance in SK.HerR cells was mediated by t-Darpp. Results: In breast tumors, PPP1R1B mRNA levels were inversely correlated with IGF-1R mRNA levels and directly associated with shorter overall survival. t-Darpp overexpression was sufficient to increase glucose metabolism in SK.tDrp cells and essential for the glycolytic phenotype of SK.HerR cells. Recombinant t-Darpp stimulated glucose uptake, glycolysis, and IGF-1R–Akt signaling in SK-BR-3 cells. Finally, t-Darpp stimulated IGF-1R heterodimerization with ErbB receptors and required IGF-1R signaling to confer its metabolic effects. Conclusions: t-Darpp activates IGF-1R signaling through heterodimerization with EGFR and HER2 to stimulate glycolysis and confer trastuzumab resistance. Clin Cancer Res; 24(5); 1216–26. ©2017 AACR.

The Signaling Pathways Project: an integrated 'omics knowledgebase for mammalian cellular signaling pathways

Public transcriptomic and ChIP-Seq datasets have the potential to illuminate facets of transcriptional regulation by mammalian cellular signaling pathways not yet explored in the research literature. Unfortunately, a variety of obstacles prevent routine re-use of these datasets by bench biologists for hypothesis generation and data validation. Here, we designed a web knowledgebase, the Signaling Pathways Project (SPP), which incorporates stable community classifications of three major categories of cellular signaling pathway node (receptors, enzymes and transcription factors) and the bioactive small molecules (BSMs) known to modulate their functions. We then subjected over 10,000 publically archived transcriptomic or ChIP-Seq experiments to a biocuration pipeline that mapped them to their relevant signaling pathway node, BSM or biosample (tissue or cell line of study). To provide for prediction of pathway node-target transcriptional regulatory relationships, we generated consensus ‘omics signatures, or consensomes, based on the significant differential expression or promoter occupancy of genomic targets across all underlying transcriptomic (expression array and RNA-Seq) or ChIP-Seq experiments. To expose the SPP knowledgebase to biology researchers, we designed a web browser interface that accommodates a variety of routine data mining strategies depending upon the requirements of the end user. Individual dataset pages provide for browsing or filtering, and facilitate integration of SPP with the research literature. Results of single gene, Gene Ontology or user-uploaded gene list queries are displayed in an interactive user interface referred to as the Regulation Report, in which evidence for transcriptional regulation of downstream genomic target by cellular signaling pathway nodes is compartmentalized in an intuitive manner. Consensome queries allow users to evaluate evidence for targets most consistently regulated by a given signaling pathway node family, and allow for detailed inspection of the pharmacology underlying node-target regulatory relationships predicted by the consensomes. Consensomes were validated using alignment with literature-based knowledge, gene target-level integration of transcriptomic and ChIP-Seq data points, and in bench experiments that confirmed previously uncharacterized node-gene target regulatory relationships. SPP is freely accessible at https://beta.signalingpathways.org. Availability and Implementation: The Signaling Pathways Project is freely accessible at https://beta.signalingpathways.org. Social media: @sigpathproject

Conserved transcriptional activity and ligand responsiveness of avian PPARs: Potential role in regulating lipid metabolism in mirgratory birds

Migratory birds undergo metabolic remodeling in tissues, including increased lipid storage in white adipose and fatty acid uptake and oxidation in skeletal muscle, to optimize energy substrate availability and utilization in preparation for long-distance flight. Different tissues undergo gene expression changes in keeping with their specialized functions and driven by tissue specific transcriptional pathways. Peroxisome proliferator-activated receptors (PPARs) are lipid-activated nuclear receptors that regulate metabolic pathways involved in lipid and glucose utilization or storage in mammals. To examine whether PPARs might mediate fatty acid activation of metabolic gene programs that would be relevant during pre-migratory fattening, we used gray catbird as the focal species. PPAR isoforms cloned from catbird share high amino acid identity with mammalian homologs (% vs human): gcPPARα (88.1%), gcPPARδ (87.3%), gcPPARγ (91.2%). We tested whether gcPPARs activated fatty acid (FA) utilization genes using Lpl and Cpt1b gene promoter-luciferase reporters in mammalian cell lines. In C2C12 mouse myocytes gcPPARα was broadly activated by the saturated and unsaturated FAs tested; while gcPPARδ showed highest activation by the mono-unsaturated FA, 18:1 oleic acid (+80%). In CV-1 monkey kidney cells gcPPARγ responded to the poly-unsaturated fatty acid, 20:5 eicosapentaenoic acid (+60%). Moreover, in agreement with their structural conservation, gcPPARs were activated by isoform selective synthetic agonists similar to the respective mammalian isoform. Adenoviral mediated over-expression of PPARα in C2C12 myocytes induced expression of genes involved in fatty acid transport, including Cd36/Fat, as well as Cpt1b, which mediates a key rate limiting step of mitochondrial β-oxidation. These gene expression changes correlated with increased lipid droplet accumulation in C2C12 myoblasts and differentiated myotubes and enhanced β-oxidation in myotubes. Collectively, the data predict that the PPARs play a conserved role in gray catbirds to regulate lipid metabolism in target tissues that undergo metabolic remodeling throughout the annual migratory cycle.

Identification of novel inverse agonists of estrogen-related receptors ERRγ and ERRβ

Estrogen-related receptors (ERRs, α, β, and γ) are orphan nuclear receptors most closely related in sequence to estrogen receptors (ERα and ERβ). Much attention has been paid recently to the functions of ERRs for their potential roles as new therapeutic targets implicated in the etiology of metabolic disorders. While no endogenous ligand has been identified for any of the ERR isoforms to date, the potential for using synthetic small molecules to modulate their activity has been demonstrated. In the present study, a series of novel inverse agonists of ERRγ and ERRβ were synthesized using regio- and stereo-specific direct substitution of triarylethylenes. These compounds were evaluated for their ability to modulate the activities of ERRs. The rational directed substitution approach and extensive SAR studies resulted in the discovery of compound 4a (DY40) as the most potent ERRγ inverse agonist described to date with mixed ERRγ/ERRβ functional activities, which potently suppressed the transcriptional functions of ERRγ with IC50=0.01μM in a cell-based reporter gene assay and antagonized ERRγ with a potency approximately 60 times greater than its analog Z-4-OHT (Z-4-hydroxytamoxifen). In addition, compound 3h (DY181) was identified as the most potent synthetic inverse agonist for the ERRβ that exhibited excellent selectivity over ERRα/γ in functional assays. This selectivity was also supported by computational docking models that suggest DY181 forms more extensive hydrogen bound network with ERRβ which should result in higher binding affinity on ERRβ over ERRγ.