Search

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Chemical platforms that facilitate both the identification and elucidation of new areas for therapeutic development are necessary but lacking. Activity-based protein profiling (ABPP) leverages active site-directed chemical probes as target discovery tools that resolve activity from expression and immediately marry the targets identified with lead compounds for drug design. However, this approach has traditionally focused on predictable and intrinsic enzyme functionality. Here, we applied our activity-based proteomics discovery platform to map non-encoded and post-translationally acquired enzyme functionalities (e.g. cofactors)in vivousing chemical probes that exploit the nucleophilic hydrazine pharmacophores found in a classic antidepressant drug (e.g. phenelzine, Nardil®). We show the probes arein vivoactive and can map proteome-wide tissue-specific target engagement of the drug. In addition to engaging targets (flavoenzymes monoamine oxidase A/B) that are associated with the known therapeutic mechanism as well as several other members of the flavoenzyme family, the probes captured the previously discoveredN-terminal glyoxylyl (Glox) group of Secernin-3 (SCRN3)in vivothrough a divergent mechanism, indicating this functional feature has biochemical activity in the brain. SCRN3 protein is ubiquitously expressed in the brain, yet gene expression is regulated by inflammatory stimuli. In an inflammatory pain mouse model, behavioral assessment of nociception showedScrn3male knockout mice selectively exhibited impaired thermal nociceptive sensitivity. Our study provides a guided workflow to entangle molecular (off)targets and pharmacological mechanisms for therapeutic development.

Ischemic heart disease is a leading cause of death in the world. Formation of new blood vessels is fundamental to cardiac reperfusion and repair after myocardial infarction (MI), and therapeutic promotion of angiogenesis, therefore, has emerged as a promising strategy for treating ischemic heart diseases. However, the therapeutic efficacy is small and transient in most of pro-angiogenic therapy. Here, we characterize unexpectedly prominent vessel aberrancy, albeit a robust angiogenesis, in MI-associated cardiac tissue by 3-dementional light sheet fluorescence imaging. Our single-cell transcriptome analysis of mouse MI tissues with Cdh5-Cre ; Rosa-LSL-tdTomato -mediated endothelial lineage tracing identifies a robust EC plasticity to acquire mesenchymal gene signature in MI-associated ECs, peaked at 1 week and partially retained at 4 and 8 weeks after MI. Moreover, cardiac ECs undergo transcriptional activation to drive mesenchymal phenotypes including over-proliferation, migration and increased permeability, which lead to vessel abnormality after exposure to in vitro ischemic microenvironment. Furthermore, our single-cell and bulk RNA-seq analysis uncovers a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, culminating in aberrant vascularization. Notably, EC-specific knockout of PDGFR-β abrogates mesenchymal gene expression in ECs, attenuates vascular abnormalities in the infarcted tissue, and improves cardiac repair after MI. Interestingly, our single-nuclei analysis uncovers that genetic ablation of PDGFR-β in ECs temporally reprograms cardiomyocyte metabolism and enhances myocyte survival and proliferation. Pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery normalizes vasculature and improves cardiac function recovery after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer exciting opportunities for normalizing vasculature and treating ischemic heart disease.

Myocardial infarction (MI) is a leading cause of death worldwide, largely because efficient interventions to restore cardiac function after MI are currently lacking. Here, we characterize vascular aberrancies induced by MI, and propose to target acquired endothelial cell (EC) changes to normalize vessels and promote cardiac repair after MI. Single-cell transcriptome analyses of MI-associated ECs indicates that ECs acquire mesenchymal gene signature that result in phenotypic and functional changes and lead to vessel abnormalities. We identify a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, altogether causing aberrant vascularization. EC-specific knockout of PDGFR-β, pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery in mice attenuates vascular abnormalities in the infarcted tissue and improves cardiac repair after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer opportunities for normalizing vasculature and treating ischemic heart diseases.

Cardiac metabolic dysfunction is a hallmark of heart failure. Estrogen related receptors ERRα and ERRγ are essential regulators for cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for heart failure. However, no natural or synthetic ERR agonist is available to demonstrate their pharmacological effectin vivo. Using a structure-based design approach, we designed and synthesized two structurally distinct pan-ERR agonists, SLU-PP-332 (332) and SLU-PP-915 (915), which significantly improved ejection fraction and ameliorated fibrosis against pressure overload-induced heart failure without affecting cardiac hypertrophy. Mechanistically, a broad-spectrum of metabolic genes were transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function, which were mainly mediated by ERRγ. Metabolomics analysis showed significant normalization of metabolic profiles in fatty acid/lipid and TCA/OXPHOS metabolites by 915 in the mouse heart with 6-week pressure overload. Autophagy was also induced by ERR agonists in cardiomycoyte. On the other hand, ERR agonism led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocyte. In summary, ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload-induced heart failurein vivo. Our results provided direct pharmacological evidence supporting the further development of ERR agonists as novel heart failure therapeuticsin vivo.

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and β are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both

The heart plays a central role in the circulatory system and provides essential oxygen, nutrients, and growth factors to the whole organism. The heart can synthesize and secrete endocrine signals to communicate with distant target organs. Studies of long-known and recently discovered heart-derived hormones highlight a shared theme and reveal a unified mechanism of heart-derived hormones in coordinating cardiac function and target organ biology. This paper reviews the biochemistry, signaling, function, regulation, and clinical significance of representative heart-derived hormones, with a focus on the cardiovascular system. This review also discusses important and exciting questions that will advance the field of cardiac endocrinology.

The human heart is vital for our survival and health, and it presents remarkable anatomical, cellular and functional heterogeneity. The four chambers of the heart, together with specialized arteries, veins, valves and conduction cells, perform distinct yet essential physiological functions. A significant gap of knowledge is that different cell's molecular signature, spatial distribution and interactions, and functional state remain little understood at the single-cell level. The goal of the heart Organ-Specific Project (OSP) is to address this knowledge gap and generate high quality, single-cell resolution, longitudinal imaging and multiomics data of normal human hearts across the entire human lifespan. To achieve this goal, we propose the following three specific aims: 1) To refine protocols of biospecimen processing, multiomics and imaging assays and define inter-individual variability using our existing banked normal human hearts. 2) To procure, archive and annotate high-quality normal heart samples across the entire human life span. We have established a streamlined procurement and biorepository infrastructure to support our heart OSP. We will procure normal heart and bone from the same donor of 5 different age groups across the entire human life span. 3) To spatially and quantitatively profile normal heart specimens across the entire human lifespan using a set of robust and scalable imaging and single-cell omics assays. In summary, the heart OSP will broadly impact the entire research community and jumpstart basic-science and medical discoveries based on a sophisticated understanding of the key molecular circuits underlying the development and aging of human heart.

Kidney disease development is poorly understood due to the complex cellular interactions of more than 25 different cell types, each with specialized functions. Here, we used single cell RNA-sequencing to resolve cell type-specific and cell fraction changes in kidney disease.Whole kidney RNA-sequencing results were strongly influenced by cell fraction changes, but minimally informative to detect cell type-specific gene expression changes. Cell type-specific differential expression analysis identified proximal tubule cells (PT cells) as the key vulnerable cell type in diseased kidneys. Through unbiased cell trajectory analyses, we show that PT cell differentiation state is altered in disease state. Lipid metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and reproducible association with PT cells state. The coupling of cell state and metabolism is established by nuclear receptors such as PPARA and ESRRA that not only control cellular metabolism but also the expression of PT cell-specific genes in mice and patient samples.

The heart plays a central role in the circulatory system and provides essential oxygen, nutrients, and growth factors to the whole organism. The heart can synthesize and secrete endocrine signals to communicate with distant target organs. Studies of long-known and recently discovered heart-derived hormones highlight a shared theme and reveal a unified mechanism of heart-derived hormones in coordinating cardiac function and target organ biology. This paper reviews the biochemistry, signaling, function, regulation, and clinical significance of representative heart-derived hormones, with a focus on the cardiovascular system. This review also discusses important and exciting questions that will advance the field of cardiac endocrinology.

Brown adipose tissue is a thermogenic organ that dissipates chemical energy as heat to protect animals against hypothermia and to counteract metabolic disease. However, the transcriptional mechanisms that determine the thermogenic capacity of brown adipose tissue before environmental cold are unknown. Here we show that histone deacetylase 3 (HDAC3) is required to activate brown adipose tissue enhancers to ensure thermogenic aptitude. Mice with brown adipose tissue-specific genetic ablation of HDAC3 become severely hypothermic and succumb to acute cold exposure. Uncoupling protein 1 (UCP1) is nearly absent in brown adipose tissue lacking HDAC3, and there is also marked downregulation of mitochondrial oxidative phosphorylation genes resulting in diminished mitochondrial respiration. Remarkably, although HDAC3 acts canonically as a transcriptional corepressor, it functions as a coactivator of oestrogen-related receptor α (ERRα) in brown adipose tissue. HDAC3 coactivation of ERRα is mediated by deacetylation of PGC-1α and is required for the transcription of Ucp1, Ppargc1a (encoding PGC-1α), and oxidative phosphorylation genes. Importantly, HDAC3 promotes the basal transcription of these genes independently of adrenergic stimulation. Thus, HDAC3 uniquely primes Ucp1 and the thermogenic transcriptional program to maintain a critical capacity for thermogenesis in brown adipose tissue that can be rapidly engaged upon exposure to dangerously cold temperature.

The endocrine system is crucial for maintaining whole‐body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than atrial/brain natriuretic peptides discovered over 30 years ago. Here, we identify growth differentiation factor 15 (GDF15) as a heart‐derived hormone that regulates body growth. We show that pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. We demonstrate that blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart‐derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive (FTT). Together these studies reveal a new endocrine mechanism by which the heart coordinates cardiac function and body growth. Our results also provide a potential mechanism for the well‐established clinical observation that children with heart diseases often develop FTT.

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell-type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-Seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-Seq overcomes the technical challenge of isolating intact single cell from complex tissues including the maturing mammalian hearts, reduces biased recovery of easily dissociated cell types and minimizes aberrant gene expression during the whole-cell dissociation. We have recently applied sNucDrop-Seq, a droplet microfluidics-based massively parallel snRNA-Seq method, to investigate the transcriptional landscape of postnatal mouse hearts in both healthy and mitochondrial disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant cellular heterogeneity in the postnatal developing heart. When applied to a mouse model of mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution. Here, we expanded these earlier studies and used our dataset to further decipher the cardiac cell type-specific gene regulatory networks. Our analysis reveals novels insights into the key nodes of gene networks that control the postnatal development and disease-associated changes of different cardiac cell types. Our ongoing work is using genetic mouse models to validate these findings from single cell analysis.

Kidney fibrosis is characterized by tubule atrophy, compromised bioenergetics and proinflammatory gene expression. The relationship between these events are poorly understood. In patient samples, the expression of TFAM and mitochondrial genes correlates with kidney fibrosis. Mice with tubule specific deletion of TFAM (Ksp-Cre/Tfam flox/flox) developed severe mitochondrial loss and decline of ATP content by 6 weeks of age. Progressive azotemia, kidney fibrosis and death of the animals was only observed after 12 weeks of age. Mechanistic studies demonstrated that aberrant mtDNA packaging upon TFAM deficiency in tubule cells resulted in escape of mtDNA into the cytosol, activation of the cytosolic DNA sensing pathway, the Stimulator of interferon genes (STING), resulting in cytokine expression and immune cell recruitment. Genetic deletion or pharmacological inhibition of STING ameliorated TFAM-loss induced kidney fibrosis, demonstrating, that in addition to its essential role in metabolism, TFAM sequesters mtDNA to prevent the activation of innate immune pathways and fibrosis.

Growth differentiation factor 15 (GDF15) is a secreted protein with pleotropic functions from the transforming growth factor β (TGF-β) family. GDF15 is synthesized as a precursor and undergoes proteolytic cleavage to generate mature GDF15. The strong appetite-suppressing effect of mature GDF15 makes it an attractive therapeutic agent/target for diseases such as obesity and cachexia. In addition, clinical studies indicate that circulating, mature GDF15 is an independent biomarker for heart failure. We recently found that GDF15 functions as a heart-derived hormone that inhibits liver growth hormone signaling and postnatal body growth in the pediatric period. However, little is known about the mechanism of GDF15 maturation, in particular the enzymes that mediate GDF15 precursor cleavage. We investigated which candidate proteases can cleave GDF15 precursor and generate mature GDF15 in cardiomyocytes in vitro and mouse hearts in vivo. We discovered that three members of the proprotein convertase, subtilisin/kexin-type (PCSK) family, namely, PCSK3, PCSK5, and PCSK6, can efficiently cleave GDF15 precursor, therefore licensing its maturation both in vitro and in vivo. Our studies suggest that PCSK3, -5, and -6 mediate a crucial step of GDF15 maturation through proteolytic cleavage of the precursor. These results also reveal new targets for therapeutic application of GDF15 in treating obesity and cachexia.

Kidney disease is poorly understood because of the organ’s cellular diversity. We used single-cell RNA sequencing not only in resolving differences in injured kidney tissue cellular composition but also in cell-type-specific gene expression in mouse models of kidney disease. This analysis highlighted major changes in cellular diversity in kidney disease, which markedly impacted whole-kidney transcriptomics outputs. Cell-type-specific differential expression analysis identified proximal tubule (PT) cells as the key vulnerable cell type. Through unbiased cell trajectory analyses, we show that PT cell differentiation is altered in kidney disease. Metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and most reproducible association with PT cell differentiation and disease. Coupling of cell differentiation and the metabolism was established by nuclear receptors (estrogen-related receptor alpha [ESRRA] and peroxisomal proliferation-activated receptor alpha [PPARA]) that directly control metabolic and PT-cell-specific gene expression in mice and patient samples while protecting from kidney disease in the mouse model.

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than ANP and BNP discovered over 30 years ago. We recently identified growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. Pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. Blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive. In addition, we used massively parallel single-nucleus RNA-Seq to decipher cardiac cell type specific gene regulatory network that induces GDF15 transcription in heart disease condition. We further determined how GDF15 precursor is processed into mature hormone. Together these studies reveal a new endocrine mechanism (cardiac endocrinology) by which the heart coordinates cardiac function and body growth.

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-seq overcomes the technical challenge of isolating intact single cells from complex tissues, including the maturing mammalian hearts; reduces biased recovery of easily dissociated cell types; and minimizes aberrant gene expression during the whole-cell dissociation. Here we applied sNucDrop-seq, a droplet microfluidics-based massively parallel snRNA-seq method, to investigate the transcriptional landscape of postnatal maturing mouse hearts in both healthy and disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant heterogeneity of cardiomyocytes, fibroblasts, and endothelial cells in postnatal developing hearts. When applied to a mouse model of pediatric mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution, including changes of subtype composition, maturation states, and functional remodeling of each cell type. Furthermore, we employed sNucDrop-seq to decipher the cardiac cell type-specific gene regulatory network (GRN) of GDF15, a heart-derived hormone and clinically important diagnostic biomarker of heart disease. Together, our results present a rich resource for studying cardiac biology and provide new insights into heart disease using an approach broadly applicable to many fields of biomedicine.

Mitochondrial dysfunction is increasingly recognized as a critical determinant of both hereditary and acquired kidney diseases. However, it remains poorly understood how mitochondrial metabolism is regulated to support normal kidney function and how its dysregulation contributes to kidney disease. Here, we show that the nuclear receptor estrogen-related receptor gamma (ERRγ) and hepatocyte nuclear factor 1 beta (HNF1β) link renal mitochondrial and reabsorptive functions through coordinated epigenomic programs. ERRγ directly regulates mitochondrial metabolism but cooperatively controls renal reabsorption via convergent binding with HNF1β. Deletion of ERRγ in renal epithelial cells (RECs), in which it is highly and specifically expressed, results in severe renal energetic and reabsorptive dysfunction and progressive renal failure that recapitulates phenotypes of animals and patients with HNF1β loss-of-function gene mutations. Moreover, ERRγ expression positively correlates with renal function and is decreased in patients with chronic kidney disease (CKD). REC-ERRγ KO mice share highly overlapping renal transcriptional signatures with human patients with CKD. Together these findings reveal a role for ERRγ in directing independent and HNF1β-integrated programs for energy production and use essential for normal renal function and the prevention of kidney disease.

The brain has the highest mitochondrial energy demand of any organ. Therefore, subtle changes in mitochondrial energy production will preferentially affect the brain. Considerable biochemical evidence has accumulated revealing mitochondrial defects associated with neuropsychiatric diseases. Moreover, the mitochondrial genome encompasses over a thousand nuclear DNA (nDNA) genes plus hundreds to thousands of copies of the maternally-inherited mitochondrial DNA (mtDNA). Therefore, partial defects in either the nDNA or mtDNA genes or combinations of the two can be sufficient to cause neuropsychiatric disorders. Inherited and acquired mtDNA mutations have recently been associated with autism spectrum disorders (ASD), which parallel previous evidence of mtDNA variation in other neurological diseases. Therefore, mitochondrial dysfunction may be central to the etiology of a wide spectrum of neurological diseases. The mitochondria and the nucleus communicate to coordinate energy production and utilization, providing the potential for therapeutics by manipulating nuclear regulation of mitochondrial gene expression.

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77⁻/⁻, or Nor1⁻/⁻ null hematopoetic precursors into LDL receptor (LDLR)⁻/⁻ recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6C(lo) monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6C(lo) monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6C(lo) monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.

Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.

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(ERR gamma), coordinates expression of genes central to ion homeostasis in oxidative tissues. Renal, gastric, and cardiac tissues subjected to genomic analysis of expression in perinatal ERR gamma null mice revealed a characteristic dysregulation of genes involved in transport processes, exemplified by the voltage-gated potassium channel, Kcne2. Consistently, ERR gamma 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 ERR gamma null mice. These findings suggest a potential role for genetic polymorphisms at the ERR gamma locus and ERR gamma modulators in the etiology and treatment of renal, gastric, and cardiac dysfunction. ( Molecular Endocrinology 24: 299-309, 2010)

OBJECTIVE Nur77 is an orphan nuclear receptor with pleotropic functions. Previous studies have identified Nur77 as a transcriptional regulator of glucose utilization genes in skeletal muscle and gluconeogenesis in liver. However, the net functional impact of these pathways is unknown. To examine the consequence of Nur77 signaling for glucose metabolism in vivo, we challenged Nur77 null mice with high-fat feeding. RESEARCH DESIGN AND METHODS Wild-type and Nur77 null mice were fed a high-fat diet (60% calories from fat) for 3 months. We determined glucose tolerance, tissue-specific insulin sensitivity, oxygen consumption, muscle and liver lipid content, muscle insulin signaling, and expression of glucose and lipid metabolism genes. RESULTS Mice with genetic deletion of Nur77 exhibited increased susceptibility to diet-induced obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp studies revealed greater high-fat diet–induced insulin resistance in both skeletal muscle and liver of Nur77 null mice compared with controls. Loss of Nur77 expression in skeletal muscle impaired insulin signaling and markedly reduced GLUT4 protein expression. Muscles lacking Nur77 also exhibited increased triglyceride content and accumulation of multiple even-chained acylcarnitine species. In the liver, Nur77 deletion led to hepatic steatosis and enhanced expression of lipogenic genes, likely reflecting the lipogenic effect of hyperinsulinemia. CONCLUSIONS Collectively, these data demonstrate that loss of Nur77 influences systemic glucose metabolism and highlight the physiological contribution of muscle Nur77 to this regulatory pathway.

The nuclear receptor corepressor, silencing mediator of retinoid and thyroid hormone receptors (SMRT), is recruited by a plethora of transcription factors to mediate lineage and signal-dependent transcriptional repression. We generated a knockin mutation in the receptor interaction domain (RID) of SMRT (SMRT mRID ) that solely disrupts its interaction with nuclear hormone receptors (NHRs). SMRT mRID mice are viable and exhibit no gross developmental abnormalities, demonstrating that the reported lethality of SMRT knockouts is determined by non-NHR transcription factors. However, SMRT mRID mice exhibit widespread metabolic defects including reduced respiration, altered insulin sensitivity, and 70% increased adiposity. The latter phenotype is illustrated by the observation that SMRT mRID -derived MEFs display a dramatically increased adipogenic capacity and accelerated differentiation rate. Collectively, our results demonstrate that SMRT-RID-dependent repression is a key determinant of the adipogenic set point as well as an integrator of glucose metabolism and whole-body metabolic homeostasis.

Innervation is important for normal metabolism in skeletal muscle, including insulin-sensitive glucose uptake. However, the transcription factors that transduce signals from the neuromuscular junction to the nucleus and affect changes in metabolic gene expression are not well defined. We demonstrate here that the orphan nuclear receptor Nur77 is a regulator of gene expression linked to glucose utilization in muscle. In vivo, Nur77 is preferentially expressed in glycolytic compared with oxidative muscle and is responsive to β-adrenergic stimulation. Denervation of rat muscle compromises expression of Nur77 in parallel with that of numerous genes linked to glucose metabolism, including glucose transporter 4 and genes involved in glycolysis, glycogenolysis, and the glycerophosphate shuttle. Ectopic expression of Nur77, either in rat muscle or in C2C12 muscle cells, induces expression of a highly overlapping set of genes, including glucose transporter 4, muscle phosphofructokinase, and glycogen phosphorylase. Furthermore, selective knockdown of Nur77 in rat muscle by small hairpin RNA or genetic deletion of Nur77 in mice reduces the expression of a battery of genes involved in skeletal muscle glucose utilization in vivo. Finally, we show that Nur77 binds the promoter regions of multiple genes involved in glucose metabolism in muscle. These results identify Nur77 as a potential mediator of neuromuscular signaling in the control of metabolic gene expression.

PPARgamma is the master regulator of adipogenesis and the molecular target of the thiazolidinedione antidiabetic drugs. By screening for compounds that promote adipogenesis, we identified a small molecule that targets the PPARgamma pathway by a distinct mechanism. This molecule, harmine, is not a ligand for the receptor; rather, it acts as a cell-type-specific regulator of PPARgamma expression. Administration of harmine to diabetic mice mimics the effects of PPARgamma ligands on adipocyte gene expression and insulin sensitivity. Unlike thiazolidinediones, however, harmine does not cause significant weight gain or hepatic lipid accumulation. Molecular studies indicate that harmine controls PPARgamma expression through inhibition of the Wnt signaling pathway. This work validates phenotypic screening of adipocytes as a promising strategy for the identification of bioactive small molecules and suggests that regulators of PPARgamma expression may represent a complementary approach to PPARgamma ligands in the treatment of insulin resistance.

Almost all cellular functions are powered by a continuous energy supply derived from cellular metabolism. However, it is little understood how cellular energy production is coordinated with diverse energy-consuming cellular functions. Here, using the cardiac muscle system, we demonstrate that nuclear receptors estrogen-related receptor α (ERRα) and ERRγ are essential transcriptional coordinators of cardiac energy production and consumption. On the one hand, ERRα and ERRγ together are vital for intact cardiomyocyte metabolism by directly controlling expression of genes important for mitochondrial functions and dynamics. On the other hand, ERRα and ERRγ influence major cardiomyocyte energy consumption functions through direct transcriptional regulation of key contraction, calcium homeostasis, and conduction genes. Mice lacking both ERRα and cardiac ERRγ develop severe bradycardia, lethal cardiomyopathy, and heart failure featuring metabolic, contractile, and conduction dysfunctions. These results illustrate that the ERR transcriptional pathway is essential to couple cellular energy metabolism with energy consumption processes in order to maintain normal cardiac function.

Viral infections and antiviral responses have been linked to several metabolic diseases, including Reye's syndrome, which is aspirin-induced hepatotoxicity in the context of a viral infection. We identify an interferon regulatory factor 3 (IRF3)–dependent but type I interferon–independent pathway that strongly inhibits the expression of retinoid X receptor α (RXRα) and suppresses the induction of its downstream target genes, including those involved in hepatic detoxification. Activation of IRF3 by viral infection in vivo greatly enhances bile acid– and aspirin-induced hepatotoxicity. Our results provide a critical link between the innate immune response and host metabolism, identifying IRF3-mediated down-regulation of RXRα as a molecular mechanism for pathogen-associated metabolic diseases.

Objective— The lungs of Abcg1 −/− mice accumulate macrophage foam cells that contain high levels of unesterified and esterified cholesterol, consistent with a role for ABCG1 in facilitating the efflux of cholesterol from macrophages to high-density lipoprotein (HDL) and other exogenous sterol acceptors. Based on these observations, we investigated whether loss of ABCG1 affects foam cell deposition in the artery wall and the development of atherosclerosis. Methods and Results— Bone marrow from wild-type or Abcg1 −/− mice was transplanted into Ldlr −/− or ApoE −/− mice. After administration of a high-fat/high-cholesterol diet, plasma and tissue lipid levels and atherosclerotic lesion size were quantified and compared. Surprisingly, transplantation of Abcg1 −/− bone marrow cells resulted in a significant reduction in lesion size in both mouse models, despite the fact that lipid levels increased in the lung, spleen, and kidney. Lesions of Ldlr −/− mice transplanted with Abcg1 −/− cells contained increased numbers of apoptotic cells. Consistent with this observation, in vitro studies demonstrated that Abcg1 −/− macrophages were more susceptible to oxidized low-density lipoprotein (ox-LDL)-dependent apoptosis than Abcg1 +/+ cells. Conclusions— Diet-induced atherosclerosis is impaired when atherosclerotic-susceptible mice are transplanted with Abcg1 −/− bone marrow. The demonstration that Abcg1 −/− macrophages undergo accelerated apoptosis provides a mechanism to explain the decrease in the atherosclerotic lesions.

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.

Members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression in inflammation and disease. Previous studies have shown that the lipid-activated receptors peroxisomal proliferator-activated receptor and liver X receptor inhibit nuclear factor-κB (NF-κB) signaling and inflammatory gene expression. We recently identified the NR4A subfamily of orphan nuclear receptors (Nur77/NR4A1, Nurr1/NR4A2, and NOR1/NR4A3) as lipopolysaccharide- and NF-κB-responsive genes in macrophages. However, the role of these transcription factors in macrophage gene expression is unknown. We demonstrate here that, in contrast to peroxisomal proliferator-activated receptor and liver X receptor, the role of NR4A receptors in macrophages is proinflammatory. Retroviral expression of Nur77 in macrophages leads to the transcriptional activation of multiple genes involved in inflammation, apoptosis, and cell cycle control. One particularly interesting Nur77-responsive gene is the inducible kinase IKKi/IKKε, an important component of the NF-κB signaling pathway. The IKKi promoter contains a functional NR4A binding site and is activated by all three NR4A receptors in transient transfection assays. Consistent with the activation of IKKi, expression of Nur77 in macrophages potentiates the induction of inflammatory gene expression in response to lipopolysaccharide. These results identify a new role for NR4A orphan nuclear receptors in the control of macrophage gene expression during inflammation.

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.

Congenital anomalies of the kidney and urinary tract (CAKUTs) are common disorders of human development affecting the renal parechyma, renal pelvis, ureter, bladder and urethra; they show evidence of shared genetic aetiology, although the molecular basis of this remains unknown in the majority of cases. Breakpoint mapping of a de novo, apparently balanced, reciprocal translocation associated with bilateral renal agenesis has implicated the gene encoding the nuclear steroid hormone receptor ESRRG as a candidate gene for CAKUT. Here we show that the Esrrg protein is detected throughout early ureteric ducts as cytoplasmic/sub-membranous staining; with nuclear localization seen in developing nephrons. In 14.5 – 16.5 dpc (days post-conception) mouse embryos, Esrrg localizes to the subset of ductal tissue within the kidney, liver and lung. The renal ductal expression becomes localized to renal papilla by 18.5 dpc. Perturbation of function was performed in embryonic mouse kidney culture using pooled siRNA to induce knock-down and a specific small-molecule agonist to induce aberrant activation of Esrrg. Both resulted in severe abnormality of early branching events of the ureteric duct. Mouse embryos with a targeted inactivation of Esrrg on both alleles (Esrrg 2/2 ) showed agenesis of the renal papilla but normal development of the cortex and remaining medulla. Taken together, these results suggest that Esrrg is required for early branching events of the ureteric duct that occur prior to the onset of nephrogenesis. These findings confirm ESRRG as a strong candidate gene for CAKUT.

Oxidized lipids and inflammatory cytokines are believed to play a causal role in atherosclerosis through the regulation of gene expression in macrophages and other cells. Previous work has implicated the nuclear receptors peroxisome proliferator-activated receptor and liver X receptor in the control of lipid-dependent gene expression and inflammation. Here we demonstrate that expression of a third group of nuclear receptors, the NR4A ligand-independent orphan receptors, is highly inducible in macrophages by diverse inflammatory stimuli. Treatment of macrophages with lipopolysaccharide (LPS), cytokines, or oxidized lipids triggers the transcriptional induction of Nur77 (NR4A1), Nurr1 (NR4A2), and NOR1 (NR4A3) expression. Several lines of evidence point to the NF-kappaB signaling pathway as a principal mediator of inducible NR4A expression in macrophages. Analysis of the murine and human Nur77 promoters revealed two highly conserved NF-kappaB response elements. Mutation of these elements inhibited LPS-dependent expression of the Nur77 promoter in transient transfection assays. Furthermore, induction of Nur77 expression by LPS was severely compromised in fibroblasts lacking the three NF-kappaB subunits, Nfkb1, c-Rel, and RelA. Consistent with its ability to be induced by oxidized lipids, Nur77 was expressed in macrophages within human atherosclerotic lesions. These results identified NR4A nuclear receptors as potential transcriptional mediators of inflammatory signals in activated macrophages.

The PGC-1 family of coactivators stimulates the activity of certain transcription factors and nuclear receptors. Transcription factors in the sterol responsive element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. We show here that high-fat feeding, which induces hyperlipidemia and atherogenesis, stimulates the expression of both PGC-1beta and SREBP1c and 1a in liver. PGC-1beta coactivates the SREBP transcription factor family and stimulates lipogenic gene expression. Further, PGC-1beta is required for SREBP-mediated lipogenic gene expression. However, unlike SREBP itself, PGC-1beta reduces fat accumulation in the liver while greatly increasing circulating triglycerides and cholesterol in VLDL particles. The stimulation of lipoprotein transport upon PGC-1beta expression is likely due to the simultaneous coactivation of the liver X receptor, LXRalpha, a nuclear hormone receptor with known roles in hepatic lipid transport. These data suggest a mechanism through which dietary saturated fats can stimulate hyperlipidemia and atherogenesis.

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

Osteoporosis, characterized by low bone mass and structural deterioration of bone tissue with an increased susceptibility to fractures, is a major public health threat to the elderly. Bone mass homeostasis in adults is maintained locally by the balance between osteoblastic bone formation and osteoclastic bone resorption. Haploinsufficiency of PPARγ, a key transcription factor implicated previously in adipogenesis, lipid metabolism, and glucose homeostasis, has now been shown to promote osteogenesis through enhanced osteoblast formation (see the related article beginning on page 846). These findings support a reciprocal relationship between the development of bone and fat, and may prompt further exploration of the PPAR pathway as a potential target for intervention in osteoporosis. Osteoclast and osteoblast: the yin and yang that control skeletal homeostasis In vertebrates, bones undergo a process of continual renewal throughout life. This process, called bone remodeling, can be viewed as a balance between osteoblastmediated bone formation and osteoclastmediated bone resorption (1). Osteoclasts are specialized cells derived from the monocyte/macrophage lineage that degrade extracellular bone matrix (2). On the other hand, mesenchyme-derived osteoblasts rebuild the resorbed bone by elaborating matrix that subsequently undergoes mineralization (3). An imbalance between the two arms of bone remodeling is associated with diseases including rheumatoid

Macrophages are an important source of angiogenic activity in wound healing, cancer, and chronic inflammation. Vascular endothelial growth factor (VEGF), a cytokine produced by macrophages, is a primary inducer of angiogenesis and neovascularization in these contexts. VEGF expression by macrophages is known to be stimulated by low oxygen tension as well as by inflammatory signals. In this study, we provide evidence that Vegfa gene expression is also regulated by activation of liver X receptors (LXRs). VEGF mRNA was induced in response to synthetic LXR agonists in murine and human primary macrophages as well as in murine adipose tissue in vivo. The effects of LXR ligands on VEGF expression were independent of hypoxia-inducible factor HIF-1alpha activation and did not require the previously characterized hypoxia response element in the VEGF promoter. Rather, LXR/retinoid X receptor heterodimers bound directly to a conserved hormone response element (LXRE) in the promoter of the murine and human Vegfa genes. Both LXRalpha and LXRbeta transactivated the VEGF promoter in transient transfection assays. Finally, we show that induction of VEGF expression by inflammatory stimuli was independent of LXRs, because these effects were preserved in LXR null macrophages. These observations identify VEGF as an LXR target gene and point to a previously unrecognized role for LXRs in vascular biology.

The nuclear receptors LXRα and LXRβ have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR −/− mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE −/− mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR −/− and apoE −/− mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages in vitro as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.