The clock-controlled nuclear receptor REV-ERBα has emerged as a critical regulator of multiple pathways involved in metabolism, development and immunity. Recent evidence has highlighted a major role for the clock in epithelial cells regulating lung inflammation, mediated by control of neutrophil chemokine expression. In this thesis, I examined the role of REV-ERBα in pulmonary immunity, using in-vivo gene targeting and nebulised lipopolysaccharide (LPS), a model for gram-negative bacterial infection, ex-vivo cell biology approaches and in vitro cell models. Initial studies of Rev-Erbα knock-out mice revealed an increase in pulmonary neutrophilia and inflammation upon aerosolised LPS challenge. Moreover, by selectively deleting the REV-ERBα DNA binding domain (DBD) in the mouse bronchial epithelium, I observed exaggerated inflammatory responses to LPS and augmented CXCL5 secretion. Interestingly, a dual deletion of REV-ERBα DBD and REV-ERBβ in mouse bronchial epithelium had a more dramatic effect on neutrophil recruitment and chemokine secretion than deletion of just the REV-ERBα DBD; in both basal and bacterial challenged conditions. Ex-vivo analysis revealed bronchial epithelial cells and macrophages both responded to novel REV-ERBα synthetic ligand GSK1362 but displayed divergent inflammatory responses in presence of this compound. Finally, I observed a striking loss of REV-ERBα protein upon pro-inflammatory challenge. Further analysis revealed this degradation was dependent on the 26S proteasome and driven by sumoylation and ubiquitination of REV-ERBα. However, by using novel REV-ERB ligand GSK1362, these post-translational modifications were blocked and the protein protected from degradation. Collectively, my results propose a new model for a central role for REV-ERBα in conferring clock control to lung neutrophilic inflammation. I have also identified a feed-forward circuit activated by inflammatory stimuli, leading to suppression of the endogenous anti-inflammatory REV-ERBα protein. Finally, I have discovered a novel mechanism for small-molecule regulation of REV-ERBα, operating via suppression of endogenous protein ubiquitination process. These observations implicate REV-ERBα as a novel therapeutic target in human inflammatory disease.

Au sein du muscle squelettique, le réticulum sarcoplasmique occupe une place essentielle dans la régulation de l’homéostasie calcique et de la contraction musculaire. En particulier, le transporteur calcique SERCA, situé à la membrane du réticulum endoplasmique permet de reconstituer le contenu calcique réticulaire suite à une contraction musculaire. Dans le muscle squelettique, l’activité de SERCA est contrôlée par un peptide inhibiteur spécifique appelé la myoréguline. Nous nous intéressons au rôle du récepteur nucléaire Rev-erb-α, un répresseur de transcription connu pour favoriser la fonction musculaire et dont l’activité peut être modulée par des ligands pharmacologiques. Nos résultats montrent que Rev-erb-α réprime l’expression de la myoréguline en se fixant sur son promoteur, ce qui a pour conséquence l’augmentation de l’activité de SERCA et la hausse du contenu calcique réticulaire. Un traitement avec un agoniste de Rev-erb-α, le SR9009, améliore l’homéostasie calcique et la contractilité musculaire de souris mdx/utr+/-, un modèle de la myopathie de Duchenne. Par ailleurs, le réticulum endoplasmique est le siège de la conformation des protéines de la voie sécrétoire. Des altérations de la conformation protéique provoquent un stress réticulaire et le déclenchement de la réponse aux protéines mal-conformées qui peut conduire jusqu’à l’apoptose. Il est décrit que le stress réticulaire est un phénomène impliqué dans l’activation de la cellule satellite musculaire suite à une blessure. Nous avons établi que Rev-erb-α, en augmentant l’interaction entre le réticulum endoplasmique et la mitochondrie accroit l’activation de la réponse aux protéines mal-conformées et l’apoptose de cellules satellites activées, ce qui pourrait impacter le potentiel de régénération musculaire. En conclusion, nous avons identifié Rev-erb-α comme un modulateur de la fonction du réticulum endoplasmique dans le muscle squelettique. Dans le futur, des thérapies ciblant spécifiquement Rev-erb-α pourraient être développées dans le cadre de pathologies musculaires chez l’Homme. / Within skeletal muscle, the sarcoplasmic reticulum plays an essential role in the regulation of calcium homeostasis and muscle contraction. In particular, the SERCA transporter, located at the membrane of the endoplasmic reticulum, by pumping calcium from cytosol from reticular lumen, allows the reticular calcium content to be reconstituted following muscle contraction. In skeletal muscle, SERCA activity is controlled by a specific inhibitory peptide called myoregulin. We are interested in the role of the nuclear receptor Rev-erb-α, a transcription repressor known to promote muscle function and whose activity can be modulated by pharmacological ligands. Our results show that Rev-erb-α represses the expression of myoregulin by binding to its promoter, which results in an increase in SERCA activity and an increase in reticular calcium content. Treatment with a Rev-erb-α agonist, SR9009, improves calcium homeostasis and muscle contractility in mdx/utr+/- mice, a model of Duchenne myopathy. In addition, the endoplasmic reticulum is the site of protein conformation of the secretory pathway. Alteration in protein conformation causes reticular stress and triggers the unfolded protein response that can lead to apoptosis. It is described that reticular stress is a phenomenon involved in the activation of skeletal muscle satellite cell following an injury. We have established that Rev-erb-α, by increasing the interaction between endoplasmic reticulum and mitochondria enhances the activation of unfolded protein response and apoptosis of activated satellite cells, which could impact the muscle regeneration capacity. In conclusion, we have identified Rev-erb-α as a modulator of endoplasmic reticulum function in skeletal muscle. In the future, specific Rev-erb-α targeting therapies may be developed for human muscle diseases.

Nuclear hormone receptors (NRs) are ligand-dependent DNA binding proteins that translate nutritional and physiological signals into gene regulation. The significance of NRs in human health and disease is underscored by the availability of drugs that targets NRs for treating several diseases. In this context, a subgroup of NR family has been proposed to regulate metabolism in a cell/tissue specific manner. The Rev-erb subgroup of NRs consists of two isoforms Rev-erb and Rev-erb. These two receptors have been shown to regulate different aspects of human physiology such as metabolism, inflammation, and circadian rhythm. Many NRs are expressed in skeletal muscle, a major mass peripheral tissue that accounts for ~40% of the total body weight and energy expenditure. This lean tissue is a major site for lipid and glucose homeostasis. Skeletal muscle express and secrete cytokines which perform autocrine and paracrine function with other tissues such as adipose. Accordingly, skeletal muscle plays important role in blood lipid profile, insulin sensitivity and progression of diseases such as type 2 diabetes and obesity. In addition, many studies have shown that NRs in skeletal muscle regulate glucose, lipid and energy homeostasis. Therefore, understanding the function of NRs in skeletal muscle provides a platform for potential new therapeutic treatments for metabolic disease. Rev-erb is expressed in skeletal muscle; however the function of this receptor in skeletal muscle metabolism has not been examined. Nevertheless, considering the importance of Rev-erb subfamily in metabolism, circadian control and the role of skeletal muscle in lipid homeostasis, the function of Rev-erb in skeletal muscle metabolism needs to be further investigated. We tested the hypothesis that Rev-erb (directly and/or indirectly) regulated the genetic programs that control lipid homeostasis in skeletal muscle. Initially, we exogenously expressed a truncated version of Rev-erb without the ligand-binding domain (Rev-erb) in vitro (in the C2C12 skeletal muscle cell culture system); and in vivo (in mouse tibialis anterior muscle. Moreover, we also attenuated Rev-erb expression in skeletal muscle cells using siRNAs targeting N-terminus and hinge regions of Rev-erb. We performed candidate based expression profiling utilizing quantitative RT-PCR analysis on the Taqman Low Density Array (TLDA) platform to identify putative downstream primary and/or secondary targets of Rev-erb action in skeletal muscle cells in the context of metabolism and muscle growth. Exogenous expression of Rev-erb in skeletal muscle cells in vitro decreased the expression of several genes involved in fatty acid/lipid absorption (including Cd36, and Fabp3 and 4). Interestingly, the mRNA encoding the master lipogenic regulator, SREBP-1c was also increased after ectopic Rev-erb expression. Moreover, we observed significant induction in the mRNAs encoding interleukin-6 and IKB that are involved in the regulation of the inflammatory cascade. Finally, we also observed the marked repression of myostatin mRNA, an important protein implicated in negative regulation of muscle hypertrophy/hyperplasia and a positive regulator of body fat accumulation. In summary, our in vitro study suggested that Rev-erb regulates genes involved in metabolism, inflammation and muscle growth. Quantitative PCR analysis that utilised the Taqman Low Density Array (TLDA) platform revealed Rev-erb siRNA expression down-regulated (in a subtle but significant manner) several genes involved in lipid/glucose homeostasis and the TGF- signalling pathway. Interestingly, genes that are involved in the myostatin and TGF- signalling pathway such as Activin A receptor type 2a (ACVR2A), Smad specific E3 ubiquitin protein ligase 1 (Smurf1), and TGF- receptor 2 (TGFBR2) were identified potential (direct and/or indirect) target of Rev-erb action in skeletal muscle cells. Moreover, genes such as Citrate Synthase (CS), V-akt murine thymoma viral oncogene homolog 2 (Akt2), Peroxisome proliferator- activated receptor- coactivator (PGC)-1 (PGC1) were also significantly modulated by Rev-erb in these analyses. The expression of two mRNAs encoding a) SREBP1c and b) IKB increased by ectopic Rev-erb expression was examined in more detail. These were selected because Rev-erb has been presumed to function as a transcriptional silencer. Secondly, we had demonstrated that in vivo expression of Rev-erb (after injection and electroporation of mouse tibialis anterior muscle) increased SREBP-1c expression, and Rev-erb siRNA studies suggested that this orphan NR was necessary for optimal SREBP-1c mRNA expression. Consequently, we tested the hypothesis that Rev-erb encodes the potential to function as a transcriptional activator in skeletal muscle. To test this hypothesis, we examined whether the SREBP1c and IKB promoters were trans-activated by co-transfected Rev-erb in skeletal muscle cells. We initially tested whether Rev-erb regulates the SREBP1c promoter. Transfection experiments showed Rev-erb expression trans-activated this promoter. This observation was in contrast to previous promoter studies showing that Rev-erb is a potent repressor of gene transcription. Therefore, we subsequently performed an experiment in which we simultaneously used the Rev-erb promoter (previously characterized to be repressed by Rev-erb) and SREBP1c promoter to examine the effect of Rev-erb expression. This experiment showed that Rev-erb repressed the activity of Rev-erb promoter, and in parallel trans-activated the SREBP1c promoter. Bioinformatics analysis identified two regions covering putative Rev-erb response elements RERE1 (-1342 to -1158) and RERE2 (-525 to -401) in the SREBP1c promoter. Chromatin immuno-precipitation assays demonstrated that Rev-erb is selectively recruited to RERE2 between nucleotide positions –525 to –401 in the promoter. Unidirectional deletion analysis of the SREBP1c promoter coupled with the analysis of mutants in the LXR response elements (of the SREBP-1c promoter) confirmed that Rev-erb mediated trans-activation of SREBP1c promoter does not function through LXR response elements. Interestingly, treatment of skeletal muscle cells with Hemin, a molecule recently proposed to function as a ligand for Rev-erbs, increased SREBP1c mRNA expression. In summary these data show that Rev-erb is a novel positive regulator of SREBP1c mRNA expression in skeletal muscle. We subsequently cloned the previously characterised human IKB promoter region spanning the potential ROR and Rev-erb binding site. Transfection experiments showed that in accordance to previously published observation, ROR trans-activated the IKB promoter. However, both Rev-erb and Rev-erb when co-transfected with the IKB promoter had minimal effects on the activity of this promoter. Studies have shown that Rev-erb functions as a competitor for ROR and block ROR mediated trans-activation of its target gene expression. Interestingly, our co-transfection experiments showed that both Rev-erb and Rev-erb blocks ROR-mediated trans-activation of IKB promoter. Together, this data suggests that Rev-erb-mediated regulation of IKB transcription in skeletal muscle cells could occur through indirect mechanisms. In conclusion, our studies have shown Rev-erb directly and indirectly regulates the expression of genes involved in metabolism, inflammation and muscle growth suggesting that Rev-erb in skeletal muscle has the potential to be exploited in a therapeutic manner.

Glucocorticoids (Gcs) are a commonly used drug to target the glucocorticoid receptor (GR). The GR has a myriad of cellular and physiological effects, however, Gcs are clinically used for the treatment of inflammatory conditions due to the potent anti-inflammatory actions of GR. The anti-inflammatory effects come with serious side effects e.g. metabolic disease. I examine the role of lipid rafts in modulating the anti-inflammatory actions of Gcs, and the role of circadian rhythms in the control of Gc side effects. I tested the role of caveolin-1 (Cav1), a constituent of membrane lipid rafts, and its role in Gc suppression of inflammation. Gene expression analysis of mouse lung tissue showed that genetic depletion of Cav1 (CAV1KO) results in increased transactivation of Gc target genes. The increased Gc action, however, does not result in an increased effect on suppression of inflammation in a model of innate immunity: aerosolised lipopolysaccharide (LPS) induced lung inflammation or in a model of adaptive immunity: Ovalbumin. CAV1KO mice were protected from LPS induced inflammation, despite increased cytokine production. This suggests a differential response to LPS in lung parenchyma and alveolar macrophages dependent on Cav1. CAV1KO results in a pro-inflammatory phenotype in macrophages, and the opposite in parenchymal tissue. These data suggest that while Cav1 is an upstream regulator of Gc response, it does not have a strong enough effect to alter the ability of GR to repress inflammation in vivo. Gc treatment results in a strong metabolic phenotype, with aberrant energy metabolism, insulin resistance and hepatosteaotosis, I investigated how this side effect interacts with circadian rhythms, another key determinant of energy metabolism. Using transcriptomics of whole lung and liver taken during the day or the night, I demonstrate that the metabolic actions of Gc in the liver can be temporally separated, whilst maintaining consistent anti-inflammatory actions in both liver and lung. This temporal gene regulation by Gc is controlled by REV-ERB, a rhythmically expressed, orphan nuclear receptor, part of the core clock machinery, via a direct interaction with GR at key regulatory DNA loci. Genetic deletion of REV-ERB protects mice from the hepatosteotosis associated with Gc treatment. Taken together, these data suggest that Gcs are regulated upstream of the receptor by the core consitutent of membrane lipid rafts; Cav1, which modulates the Gc response in vivo. Also, that the GR action can be controlled by dosing at different times of day, separating the detrimental metabolic effects of Gcs from the beneficial anti-inflammatory effects. This is enabled through a direct interaction between GR and REV-ERB at key gene regulatory sites.

La première partie de mon travail de thèse a été de définir le rôle joué par le récepteur nucléaire Rev-erb alpha dans les mécanismes de synchronisation par la nourriture d’une horloge circadienne putative, non encore localisée, appelée « horloge alimentaire ». La seconde partie de mon travail a consisté à étudier la participation de Rev-erb alpha dans les régulations des métabolismes glucidique et lipidique. L’ensemble de nos données indique que le répresseur transcriptionnel Rev-erb alpha joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d’un point de vue circadien, l’absence de Rev-erb alpha altère la synchronisation à l’heure des repas – démontré par une réduction des sorties comportementales et physiologiques de l’horloge alimentaire, ainsi que par l’absence d’ajustement du rythme de la protéine d’horloge PER2 dans l’oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipides – démontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu’une perte de contrôle de l’expression de la Lipoprotéine lipase. / The work performed during this PhD thesis aimed at investigating the role of the transcriptional silencer Rev-erbα in both the circadian clockwork of the food-entrainable oscillator and metabolic regulations. Firstly, by evaluating food-anticipatory components in animals fed once a day at the same time, we showed that mice lacking Rev-erbα display a reduction in locomotor activity prior to food access compared to littermate controls. Accordingly, the rises in body temperature and corticosterone that anticipate mealtime are also diminished. Interestingly, daily p-ERK expression in hypothalamic regions and daily PER2 expression in the cerebellum of Rev-erbα KO mice are not phase-adjusted to feeding time. These results indicate that Rev-erbα participates in the integration of feeding signals and in food-seeking behaviors. Secondly, by investigating energy balance in fasted, normal chow or high-fat fed animals, we revealed that Rev-erbα KO mice exhibit greater reliance on lipid fuels as energy substrates, contributing to a mild hyperglycemic state. We also found that Lipoprotein lipase (Lpl) expression, is strongly up-regulated in peripheral tissues of Rev-erbα KO mice, predisposing mice to obesity. In this regard, we uncovered a new molecular pathway that ties clock-driven Lpl expression to energy homeostasis. These findings highlight the significance of daily Rev-erbα oscillations to prevent the appearance of the metabolic syndrome.In conclusion, we provide evidence that REV-ERBα may be a part of the food-entrainable oscillator clockwork that triggers food-anticipatory components, and represents a pivotal player to link the core clock machinery to metabolic pathways.

The nuclear receptor superfamily is responsible for regulating the expression of genes involved in development, reproduction and metabolism. These transcription factors control the expression of their target genes through the binding of small molecule regulators to their ligand binding domains. Classical nuclear receptors include the steroid receptors, which bind endocrine hormones and have been important targets of pharmaceutical intervention. However, approximately one half of the human nuclear receptors remain orphans and are without known cognate ligands. Focusing on the Drosophila orthologues of these orphan receptors, this project used mass spectrometry to identify the chemical diversity associated with the receptors following expression in recombinant systems. In a genome-wide screen of Drosophila nuclear receptors, this approach identified co-purifying molecules with a number of receptors. The physiological relevance of these putative ligand/receptor pairs was determined through biochemical analysis, in vivo characterization and structure determination. Ligand(s) or the ligand state was identified for the Drosophila receptors: DHR3, DHR96, E75, Ftz-f1 and USP. Of these, three were validated through the efforts of this project, and independent groups confirmed the remaining two. The most significant findings were the discoveries that the fly nuclear receptor E75 is regulated by heme, gas and redox, and that there is a similar regulatory scheme in the human orthologues, Reverbα and β. Furthermore, crystallization of the heme-bound Rev-erbβ ligand binding domain was also achieved, and this provided key insights into the mechanism of ligand regulation for the Rev-erbs. This project highlighted the role of nuclear receptors in metabolic surveillance. The ligands/signals identified in association with these receptors include: cholesterol, dehydrocholesterol, heme, NO, CO, redox and phospholipids. Unlike the classical steroid hormones, these are not dedicated signaling molecules, but instead are key substrates or products of metabolism. In the context of nuclear receptor signaling, I hypothesize that these metabolites serve as metabolic indicators/signals in the regulation of development and metabolism. Furthermore, four of these Drosophila receptors comprise the ecdysone-response pathway in the developing fly. Taken together, this suggests that both the metabolic state of the organism and steroid hormones drive nuclear receptor regulation of development.

The nuclear receptor superfamily is responsible for regulating the expression of genes involved in development, reproduction and metabolism. These transcription factors control the expression of their target genes through the binding of small molecule regulators to their ligand binding domains. Classical nuclear receptors include the steroid receptors, which bind endocrine hormones and have been important targets of pharmaceutical intervention. However, approximately one half of the human nuclear receptors remain orphans and are without known cognate ligands. Focusing on the Drosophila orthologues of these orphan receptors, this project used mass spectrometry to identify the chemical diversity associated with the receptors following expression in recombinant systems. In a genome-wide screen of Drosophila nuclear receptors, this approach identified co-purifying molecules with a number of receptors. The physiological relevance of these putative ligand/receptor pairs was determined through biochemical analysis, in vivo characterization and structure determination. Ligand(s) or the ligand state was identified for the Drosophila receptors: DHR3, DHR96, E75, Ftz-f1 and USP. Of these, three were validated through the efforts of this project, and independent groups confirmed the remaining two. The most significant findings were the discoveries that the fly nuclear receptor E75 is regulated by heme, gas and redox, and that there is a similar regulatory scheme in the human orthologues, Reverbα and β. Furthermore, crystallization of the heme-bound Rev-erbβ ligand binding domain was also achieved, and this provided key insights into the mechanism of ligand regulation for the Rev-erbs. This project highlighted the role of nuclear receptors in metabolic surveillance. The ligands/signals identified in association with these receptors include: cholesterol, dehydrocholesterol, heme, NO, CO, redox and phospholipids. Unlike the classical steroid hormones, these are not dedicated signaling molecules, but instead are key substrates or products of metabolism. In the context of nuclear receptor signaling, I hypothesize that these metabolites serve as metabolic indicators/signals in the regulation of development and metabolism. Furthermore, four of these Drosophila receptors comprise the ecdysone-response pathway in the developing fly. Taken together, this suggests that both the metabolic state of the organism and steroid hormones drive nuclear receptor regulation of development.

La première partie de mon travail de thèse a été de définir le rôle joué par le récepteur nucléaire Rev-erb alpha dans les mécanismes de synchronisation par la nourriture d'une horloge circadienne putative, non encore localisée, appelée " horloge alimentaire ". La seconde partie de mon travail a consisté à étudier la participation de Rev-erb alpha dans les régulations des métabolismes glucidique et lipidique. L'ensemble de nos données indique que le répresseur transcriptionnel Rev-erb alpha joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d'un point de vue circadien, l'absence de Rev-erb alpha altère la synchronisation à l'heure des repas - démontré par une réduction des sorties comportementales et physiologiques de l'horloge alimentaire, ainsi que par l'absence d'ajustement du rythme de la protéine d'horloge PER2 dans l'oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipides - démontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu'une perte de contrôle de l'expression de la Lipoprotéine lipase.