Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-γ has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-γ and RXR-α as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-γ and RXR-α form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-γ to contact multiple domains in both proteins. Three interfaces link PPAR-γ and RXR-α, including some that are DNA dependent. The PPAR-γ LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

The nuclear receptors REV-ERBα (encoded by NR1D1) and REV-ERBβ (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.REV-ERBα was originally identified as an orphan member of the nuclear hormone receptor (NHR) family on the basis of its canonical domain structure and sequence conservation 1,2 . REV-ERBβ was subsequently identified by its homology to other NHRs and its pattern of expression, which overlaps greatly with that of REV-ERBα. Both receptors have particularly high expression in the liver, adipose tissue, skeletal muscle and brain 3-8 , where they are transcribed in a circadian manner 9-11 . The REV-ERBs are unique in the NHR superfamily in that they lack the carboxy-terminal tail (helix 12) of the ligand-binding domain (LBD), which is required for coactivator recognition 12

Peripheral arterial disease (PAD) affects 5 million people in the US and is the primary cause of limb amputations. Exercise remains the single best intervention for PAD, in part thought to be mediated by increases in capillary density. How exercise triggers angiogenesis is not known. PPAR␥ coactivator (PGC)-1␣ is a potent transcriptional coactivator that regulates oxidative metabolism in a variety of tissues. We show here that PGC-1␣ mediates exercise-induced angiogenesis. Voluntary exercise induced robust angiogenesis in mouse skeletal muscle. Mice lacking PGC-1␣ in skeletal muscle failed to increase capillary density in response to exercise. Exercise strongly induced expression of PGC-1␣ from an alternate promoter. The induction of PGC-1␣ depended on ␤-adrenergic signaling. ␤-adrenergic stimulation also induced a broad program of angiogenic factors, including vascular endothelial growth factor (VEGF). This induction required PGC-1␣. The orphan nuclear receptor ERR␣ mediated the induction of VEGF by PGC-1␣, and mice lacking ERR␣ also failed to increase vascular density after exercise. These data demonstrate that ␤-adrenergic stimulation of a PGC-1␣/ERR␣/VEGF axis mediates exercise-induced angiogenesis in skeletal muscle.VEGF ͉ ERR␣ ͉ ␤-adrenergic

Rationale The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. Objective We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. Methods and Results We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3′-untranslated region, an effect that decreased expression of Id1 and increased p21WAF/CIP and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti–miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. Conclusions These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Rationale Mechanisms of angiogenesis in skeletal muscle remain poorly understood. Efforts to induce physiological angiogenesis hold promise for the treatment of diabetic microvascular disease and Peripheral Artery Disease (PAD), but are hindered by the complexity of physiological angiogenesis and by the poor angiogenic response of aged and diabetic patients. To date, the best therapy for diabetic vascular disease remains exercise, often a challenging option for patients with leg pain. PGC-1α, a powerful regulator of metabolism, mediates exercise-induced angiogenesis in skeletal muscle. Objective To test if, and how, PGC-1α can induce functional angiogenesis in adult skeletal muscle. Methods and Results We show here that muscle PGC-1α robustly induces functional angiogenesis in adult, aged, and diabetic mice. The process involves the orchestration of numerous cell types, and leads to patent, non-leaky, properly organized, and functional nascent vessels. These findings contrast sharply with the disorganized vasculature elicited by induction of VEGF alone. Bioinformatic analyses revealed that PGC-1α induces the secretion of secreted phosphoprotein 1 (SPP1), and the recruitment of macrophages. SPP1 stimulates macrophages to secrete monocyte chemoattractant protein-1 (MCP-1), which then activates adjacent endothelial cells, pericytes, and smooth muscle cells. In contrast, induction of PGC-1α in SPP1 −/− mice leads to immature capillarization and blunted arteriolarization. Finally, adenoviral delivery of PGC-1α into skeletal muscle of either young or old and diabetic mice improved the recovery of blood flow in the murine hind-limb ischemia model of PAD. Conclusions PGC-1α drives functional angiogenesis in skeletal muscle and likely recapitulates the complex physiological angiogenesis elicited by exercise.