George E. O. Muscat

Sox18 induces development of the lymphatic vasculature in mice

The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia. However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia, suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18-null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.

The NR4A subgroup: immediate early response genes with pleiotropic physiological roles.

The nuclear hormone receptor (NR) superfamily includes the orphan NR4A subgroup, comprised of Nur77 (NR4A1), Nurr1 (NR4A2) and NOR-1 (NR4A3). These NRs are classified as early response genes, are induced by a diverse range of signals, including fatty acids, stress, growth factors, cytokines, peptide hormones, phorbol esters, neurotransmitters, and physical stimuli (for example magnetic fields, shear stress). The ability to sense and rapidly respond to changes in the cellular environment thus appears to be a hallmark of this subfamily. The members of the NR4A subgroup are well conserved in the DNA binding domain (~91-95%) and the C-terminal ligand-binding domain (~60%), but are divergent in the N-terminal AB region. These receptors bind as monomers, homodimers and heterodimers with RXRs (to mediate retinoid signaling) to different permutations of the canonical NR binding motif. The NR4A subgroup activates gene expression in a constitutive ligand-independent manner. NR4A-mediated trans-activation (LBD) involves unusually active N-terminal AF-1 domains that mediate coactivator recruitment. Moreover, the NR4A receptors encode atypical LBDs and AF-2 domains. For example, the LBDs contain no cavity due to bulky hydrophobic residue side chains, and lack the classical coactivator-binding cleft constituted by helices 3, 4 and 12. However, a hydrophobic patch exists between helices 11 and 12, that encodes a novel cofactor interface that modulates transcriptional activity. In line with the pleiotropic physiological stimuli that induce the NR4A subgroup, these orphan NRs have been implicated in cell cycle regulation (and apoptosis), neurological disease, steroidogenesis, inflammation, carcinogenesis and atherogenesis.

Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis

We describe a functional and biochemical link between the myogenic activator MyoD, the deacetylase HDAC1, and the tumor suppressor pRb. Interaction of MyoD with HDAC1 in undifferentiated myoblasts mediates repression of muscle-specific gene expression. Prodifferentiation cues, mimicked by serum removal, induce both downregulation of HDAC1 protein and pRb hypophosphorylation. Dephosphorylation of pRb promotes the formation of pRb-HDAC1 complex in differentiated myotubes. pRb-HDAC1 association coincides with disassembling of MyoD-HDAC1 complex, transcriptional activation of muscle-restricted genes, and cellular differentiation of skeletal myoblasts. A single point mutation introduced in the HDAC1 binding domain of pRb compromises its ability to disrupt MyoD-HDAC1 interaction and to promote muscle gene expression. These results suggest that reduced expression of HDAC1 accompanied by its redistribution in alternative nuclear protein complexes is critical for terminal differentiation of skeletal muscle cells.

Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice.

Analysis of classical mouse mutations has been useful in the identification and study of many genes. We previously mapped Sox18, encoding an SRY-related transcription factor, to distal mouse chromosome 2 (ref. 2). This region contains a known mouse mutation, ragged (Ra), that affects the coat and vasculature. Here we have directly evaluated Sox18 as a candidate for Ra. We found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. Furthermore, we found no recombination between Sox18 and Ra in an interspecific backcross segregating for the Ra phenotype. We found point mutations in Sox18 in two different Ra alleles that result in missense translation and premature truncation of the encoded protein. Fusion proteins containing these mutations lack the ability to activate transcription relative to wild-type controls in an in vitro assay. Our observations implicate mutations in Sox18 as the underlying cause of the Ra phenotype, and identify Sox18 as a critical gene for cardiovascular and hair follicle formation.

Minireview: Nuclear hormone receptor 4A signaling: Implications for metabolic disease

Numerous members of the nuclear hormone receptor (NR) superfamily have been demonstrated to regulate metabolic function in a cell- and tissue-specific manner. This review brings together recent studies that have associated members of the NR superfamily, the orphan NR4A subgroup, with the regulation of metabolic function and disease. The orphan NR4A subgroup includes Nur77 (NR4A1), Nurr1 (NR4A2), and Nor-1 (NR4A3). Expression of these receptors is induced in multiple tissues by a diverse range of stimuli, including stimuli associated with metabolic function, such as: β-adrenoceptor agonists, cold, fatty acids, glucose, insulin, cholesterol, and thiazolidinediones. In vitro and in vivo gain- and loss-of-function studies in major metabolic tissues (including skeletal muscle, adipose, and liver cells and tissues) have associated the NR4A subgroup with specific aspects of lipid, carbohydrate, and energy homeostasis. Most excitingly, although these orphan receptors do not have known endogenous ligands, several small molecule agonists have recently been identified. The preliminary studies reviewed in this manuscript suggest that therapeutic exploitation of the NR4A subgroup may show utility against dyslipidemia, obesity, diabetes, and cardiovascular disease.

International Union of Pharmacology. LXVI. Orphan nuclear receptors.

Half of the members of the nuclear receptors superfamily are so-called “orphan” receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.

Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes

ABSTRACT In this report, we investigate the role of the RNA-binding protein HuR during skeletal myogenesis. At the onset of myogenesis in differentiating C2C12 myocytes and in vivo in regenerating mouse muscle, HuR cytoplasmic abundance increased dramatically, returning to a predominantly nuclear presence upon completion of myogenesis. mRNAs encoding key regulators of myogenesis-specific transcription (myogenin and MyoD) and cell cycle withdrawal (p21), bearing AU-rich regions, were found to be targets of HuR in a differentiation-dependent manner. Accordingly, mRNA half-lives were highest during differentiation, declining when differentiation was completed. Importantly, HuR-overexpressing C2C12 cells displayed increased target mRNA expression and half-life and underwent precocious differentiation. Our findings underscore a critical function for HuR during skeletal myogenesis linked to HuRs coordinate regulation of muscle differentiation genes.

The Orphan Nuclear Receptor, RORα, Regulates Gene Expression That Controls Lipid Metabolism STAGGERER (SG/SG) MICE ARE RESISTANT TO DIET-INDUCED OBESITY

Homozygous staggerer mice (sg/sg) display decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORalpha) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in sg/sg mice. Moreover, the sg/sg mice were characterized by reduced adiposity (associated with decreased fat pad mass and adipocyte size). Candidate-based expression profiling demonstrated that the dyslipidemia in sg/sg mice is associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This is consistent with the reduced serum lipids. The molecular mechanism did not involve aberrant expression of LXR and/or ChREBP. However, ChIP and transfection analyses revealed that RORalpha is recruited to and regulates the activity of the SREBP-1c promoter. Furthermore, the lean phenotype in sg/sg mice is also characterized by significantly increased expression of PGC-1alpha, PGC-1beta, and lipin1 mRNA in liver and white and brown adipose tissue from sg/sg mice. In addition, we observed a significant 4-fold increase in beta(2)-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORalpha expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type but not sg/sg mice exhibited a approximately 20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues are involved in decreased adiposity and resistance to diet-induced obesity in the sg/sg mice, despite hyperphagia. In conclusion, we suggest this orphan nuclear receptor is a key modulator of fat accumulation and that selective ROR modulators may have utility in the treatment of obesity.Homozygous staggerer mice (sg/sg) display decreased and dysfunctional retinoic acid receptor-related orphan receptor α (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in sg/sg mice. Moreover, the sg/sg mice were characterized by reduced adiposity (associated with decreased fat pad mass and adipocyte size). Candidate-based expression profiling demonstrated that the dyslipidemia in sg/sg mice is associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This is consistent with the reduced serum lipids. The molecular mechanism did not involve aberrant expression of LXR and/or ChREBP. However, ChIP and transfection analyses revealed that RORα is recruited to and regulates the activity of the SREBP-1c promoter. Furthermore, the lean phenotype in sg/sg mice is also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1 mRNA in liver and white and brown adipose tissue from sg/sg mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type but not sg/sg mice exhibited a ∼20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues are involved in decreased adiposity and resistance to diet-induced obesity in the sg/sg mice, despite hyperphagia. In conclusion, we suggest this orphan nuclear receptor is a key modulator of fat accumulation and that selective ROR modulators may have utility in the treatment of obesity.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for ∼40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces β-adrenergic receptor (β-AR)-mediated adaptive thermogenesis. β-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30–60 min of β-AR agonist (isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (>100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase γ3, UCP3, CD36, adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and β-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.

The activation function-1 domain of Nur77/NR4A1 mediates trans-activation, cell specificity, and coactivator recruitment

Nur77/NR4A1 is an “orphan member” of the nuclear hormone receptor superfamily. Nur77 and its close relatives Nurr1 and NOR-1 bind as monomers to a consensus binding site, the nerve growth factor induced protein I-B (NGFI-B)-binding response element (NBRE). The Nur77/NURR1/NOR1 nuclear receptors are classified as immediate early response genes which are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, and apoptosis. However, the mechanism of coactivation and ligand independent trans-activation remains unclear. Hence we examined the molecular basis of Nur77-mediated cofactor recruitment and activation. We observed that Nur77 trans-activates gene expression in a cell-specific manner, and operates in an activation function-1 (AF-1)-dependent manner. The AB region encodes an uncommonly potent N-terminal AF-1 domain delimited to between amino acids 50 and 160 and is essential for the ligand-independent activation of gene expression. Steroid receptor coactivator-2 (SRC-2) modulates the activity of the N-terminal AF-1 domain. Moreover, SRC-2 dramatically potentiates the retinoid induced RXR-dependent activation of the Nur77 ligand binding domain (LBD). Interestingly, the N-terminal AB region (not the LBD) facilitates coactivator recruitment and directly interacts with SRC, p300, PCAF, and DRIP-205. Consistent with this, homology modeling indicated that the Nur77 LBD coactivator binding cleft was substantially different from that of retinoic acid receptor γ, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a much more hydrophilic surface with a distinct topology. This observation accounts for the inability of this nuclear receptor LBD to directly mediate cofactor recruitment. Furthermore, the AF-1 domain physically associates with the Nur77 C-terminal LBD and synergizes with the retinoid X receptor LBD. Thus, the AF-1 domain plays a major role in Nur77-mediated transcriptional activation, cofactor recruitment, and intra- and intermolecular interactions.