Michael Downes

Anatomical Profiling of Nuclear Receptor Expression Reveals a Hierarchical Transcriptional Network

In multicellular organisms, the ability to regulate reproduction, development, and nutrient utilization coincided with the evolution of nuclear receptors (NRs), transcription factors that utilize lipophilic ligands to mediate their function. Studying the expression profile of NRs offers a simple, powerful way to obtain highly relational information about their physiologic functions as individual proteins and as a superfamily. We surveyed the expression of all 49 mouse NR mRNAs in 39 tissues, representing diverse anatomical systems. The resulting data set uncovers several NR clades whose patterns of expression indicate their ability to coordinate the transcriptional programs necessary to affect distinct physiologic pathways. Remarkably, this regulatory network divides along the following two physiologic paradigms: (1) reproduction, development, and growth and (2) nutrient uptake, metabolism, and excretion. These data reveal a hierarchical transcriptional circuitry that extends beyond individual tissues to form a meganetwork governing physiology on an organismal scale.

A Chemical, Genetic, and Structural Analysis of the Nuclear Bile Acid Receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.

PPARδ regulates multiple proinflammatory pathways to suppress atherosclerosis

Lipid homeostasis and inflammation are key determinants in atherogenesis, exemplified by the requirement of lipid-laden, foam cell macrophages for atherosclerotic lesion formation. Although the nuclear receptor PPARδ has been implicated in both systemic lipid metabolism and macrophage inflammation, its role as a therapeutic target in vascular disease is unclear. We show here that orally active PPARδ agonists significantly reduce atherosclerosis in apoE−/− mice. Metabolic and gene expression studies reveal that PPARδ attenuates lesion progression through its HDL-raising effect and anti-inflammatory activity within the vessel wall, where it suppresses chemoattractant signaling by down-regulation of chemokines. Activation of PPARδ also induces the expression of regulator of G protein signaling (RGS) genes, which are implicated in blocking the signal transduction of chemokine receptors. Consistent with this, PPARδ ligands repress monocyte transmigration and macrophage inflammatory responses elicited by atherogenic cytokines. These results reveal that PPARδ antagonizes multiple proinflammatory pathways and suggest PPARδ-selective drugs as candidate therapeutics for atherosclerosis.

Discovery and optimization of non-steroidal FXR agonists from natural product-like libraries

The efficient regulation of cholesterol biosynthesis, metabolism, acquisition, and transport is an essential component of lipid homeostasis. The farnesoid X receptor (FXR) is a transcriptional sensor for bile acids, the primary product of cholesterol metabolism. Accordingly, the development of potent, selective, small molecule agonists, partial agonists, and antagonists of FXR would be an important step in further deconvoluting FXR physiology. Herein, we describe the development of four novel classes of potent FXR activators originating from natural product-like libraries. Initial screening of a 10,000-membered, diversity-orientated library of benzopyran containing small molecules for FXR activation utilizing a cell-based reporter assay led to the identification of several lead compounds possessing low micromolar activity (EC50s = 5-10 microM). These compounds were systematically optimized employing parallel solution-phase synthesis and solid-phase synthesis to provide four classes of compounds that potently activate FXR. Two series of compounds, bearing stilbene or biaryl moieties, contain members that are the most potent FXR agonists reported to date in cell-based assays. These compounds may find future utility as chemical tools in studies aimed at further defining the physiological role of FXR and discovering potential therapeutic agents for the treatment of diseases linked to cholesterol and bile acid metabolism and homeostasis.

ERRγ Regulates Cardiac, Gastric, and Renal Potassium Homeostasis

Energy production by oxidative metabolism in kidney, stomach, and heart, is primarily expended in establishing ion gradients to drive renal electrolyte homeostasis, gastric acid secretion, and cardiac muscle contraction, respectively. In addition to orchestrating transcriptional control of oxidative metabolism, the orphan nuclear receptor, estrogen-related receptor gamma (ERRgamma), coordinates expression of genes central to ion homeostasis in oxidative tissues. Renal, gastric, and cardiac tissues subjected to genomic analysis of expression in perinatal ERRgamma null mice revealed a characteristic dysregulation of genes involved in transport processes, exemplified by the voltage-gated potassium channel, Kcne2. Consistently, ERRgamma null animals die during the first 72 h of life with elevated serum potassium, reductions in key gastric acid production markers, and cardiac arrhythmia with prolonged QT intervals. In addition, we find altered expression of several genes associated with hypertension in ERRgamma null mice. These findings suggest a potential role for genetic polymorphisms at the ERRgamma locus and ERRgamma modulators in the etiology and treatment of renal, gastric, and cardiac dysfunction.

Transcriptional repression by COUP-TF II is dependent on the C-terminal domain and involves the N-CoR variant, RIP13δ1

COUP-TF II/ARP-1 is an orphan steroid receptor that inhibits basal transcription, and represses trans-activation by the vitamin D, thyroid hormone and retinoid receptors. The molecular basis of repression by COUP-TF II remains obscure. In this study we utilized the GAL4 hybrid system to demonstrate that COUP-TF II contains sequences within the C-terminal region that encode a dominant transcriptional repressor that inhibits the ability of the potent chimeric transactivator GAL4VP16 to induce transcription. Mammalian two hybrid analysis demonstrated that COUP-TF II did not efficiently interact with either interaction domains I or II from N-CoR and RIP13. However, COUP-TF II efficiently interacts with a region comprised of interaction domains I + II from the corepressor, RIP13delta1. Efficient interaction of the orphan receptor with the corepressor was critically dependent on a large region comprised of the C, D and E domains of COUP-TF II, which correlated with the domain that maximally represses transcription. This investigation suggested that the N-CoR variant, RIP13delta1 interacts with a region of COUP-TF II that functions as a dominant transcriptional repressor.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2–3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Nuclear Receptors and AMPK: Resetting Metabolism

Obesity, and in particular central adiposity, is a key feature of metabolic syndrome, which includes trends toward increased triglycerides, insulin resistance, high blood pressure, hypercholesterolemia, and heart disease. It has a prevalence of 25% or more and is a dominant component of the health care budgets in Western societies. In addition to genetic causes, high-fat diets and disrupted sleep patterns have major influences on the development of metabolic syndrome. Recent studies have demonstrated active roles for the nuclear receptor superfamily and the energy-sensing kinase adenosine monophosphate (AMP)-activated protein kinase (AMPK) in regulating metabolism and circadian rhythm. In this chapter, we review these findings and attempt to develop a better understanding of the interplay between metabolism and circadian rhythm and their coordinated regulation by nuclear receptors and AMPK. This supraregulatory network may be considered a target for novel therapeutic applications against metabolic syndrome.

Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P<E−100 for all. Of the nearly 11u2009000 VDR-binding peaks identified across the genome in DC1s, 1317 were shared with DC2s (91% of DC2 sites) and 1579 with monocytes (83% of monocyte sites). Latitude-dependent autoimmune disease risk polymorphisms were highly overrepresented within 5u2009kb of the peaks. Several transcription factor recognition motifs were highly overrepresented in the peaks, including those for the autoimmune risk gene, BATF. This evidence indicates that VDR regulates hundreds of myeloid cell genes and that the molecular pathways controlled by VDR in these cells are important in maintaining tolerance.

Intestinal PPARδ protects against diet-induced obesity, insulin resistance and dyslipidemia

Peroxisome proliferator-activated receptor δ (PPARδ) is a ligand-activated transcription factor that has an important role in lipid metabolism. Activation of PPARδ stimulates fatty acid oxidation in adipose tissue and skeletal muscle and improves dyslipidemia in mice and humans. PPARδ is highly expressed in the intestinal tract but its physiological function in this organ is not known. Using mice with an intestinal epithelial cell-specific deletion of PPARδ, we show that intestinal PPARδ protects against diet-induced obesity, insulin resistance and dyslipidemia. Furthermore, absence of intestinal PPARδ abolished the ability of PPARδ agonist GW501516 to increase plasma levels of HDL-cholesterol. Together, our findings show that intestinal PPARδ is important in maintaining metabolic homeostasis and suggest that intestinal-specific activation of PPARδ could be a therapeutic approach for treatment of the metabolic syndrome and dyslipidemia, while avoiding systemic toxicity.