The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

BACKGROUND: The circadian clock plays a crucial role in regulating physiology and is important for maintaining immune homeostasis and responses to inflammatory stimuli. Inflammatory arthritis often shows diurnal variation in disease symptoms and disease markers, and it is now established that cellular clocks regulate joint inflammation. The clock gene Bmal1 is critical for maintenance of 24-h rhythms and plays a key role in regulating immune responses, as well as in aging-related processes. Fibroblast-like synoviocytes (FLS) are circadian rhythmic joint mesenchymal cells which are important for maintenance of joint health and play a crucial role in the development of inflammatory arthritis. The aim of this study was to investigate the importance of the joint mesenchymal cell circadian clock in health and disease. METHODS: Mice were generated which lack Bmal1 in Col6a1-expressing cells, targeting mesenchymal cells in the ankle joints. Joints of these animals were assessed by X-ray imaging, whole-mount staining and histology, and the composition of the synovium was assessed by flow cytometry. Arthritis was induced using collagen antibodies. RESULTS: Bmal1 deletion in joint mesenchymal cells rendered the FLS and articular cartilage cells arrhythmic. Targeted mice exhibited significant changes in the architecture of the joints, including chondroid metaplasia (suggesting a switch of connective tissue stem cells towards a chondroid phenotype), reductions in resident synovial macrophages and changes in the basal pro-inflammatory activity of FLS. Loss of Bmal1 in FLS rendered these resident immune cells more pro-inflammatory in response to challenge, leading to increased paw swelling, localised infiltration of mononuclear cells and enhanced cytokine production in a model of arthritis. CONCLUSIONS: This study demonstrates the importance of Bmal1 in joint mesenchymal cells in regulating FLS and chondrocyte development. Additionally, we have identified a role for this core clock component for restraining local responses to inflammation and highlight a role for the circadian clock in regulating inflammatory arthritis.

The circadian clock regulates inflammatory arthritis.

There is strong diurnal variation in the symptoms and severity of chronic inflammatory diseases, such as rheumatoid arthritis. In addition, disruption of the circadian clock is an aggravating factor associated with a range of human inflammatory diseases. To investigate mechanistic links between the biological clock and pathways underlying inflammatory arthritis, mice were administered collagen (or saline as a control) to induce arthritis. The treatment provoked an inflammatory response within the limbs, which showed robust daily variation in paw swelling and inflammatory cytokine expression. Inflammatory markers were significantly repressed during the dark phase. Further work demonstrated an active molecular clock within the inflamed limbs and highlighted the resident inflammatory cells, fibroblast-like synoviocytes (FLSs), as a potential source of the rhythmic inflammatory signal. Exposure of mice to constant light disrupted the clock in peripheral tissues, causing loss of the nighttime repression of local inflammation. Finally, the results show that the core clock proteins cryptochrome (CRY) 1 and 2 repressed inflammation within the FLSs, and provide novel evidence that a CRY activator has anti-inflammatory properties in human cells. We conclude that under chronic inflammatory conditions, the clock actively represses inflammatory pathways during the dark phase. This interaction has exciting potential as a therapeutic avenue for treatment of inflammatory disease.-Hand, L. E., Hopwood, T. W., Dickson, S. H., Walker, A. L., Loudon, A. S. I., Ray, D. W., Bechtold, D. A., Gibbs, J. E. The circadian clock regulates inflammatory arthritis.

Adiponectin induces A20 expression in adipose tissue to confer metabolic benefit.

Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα(-/-) mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3ß activation and induction of A20. Attenuated inflammatory responses in Reverbα(-/-) WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery-induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.

Induction of the metabolic regulator Txnip in fasting-induced and natural torpor.

Torpor is a physiological state characterized by controlled lowering of metabolic rate and core body temperature, allowing substantial energy savings during periods of reduced food availability or harsh environmental conditions. The hypothalamus coordinates energy homeostasis and thermoregulation and plays a key role in directing torpor. We recently showed that mice lacking the orphan G protein-coupled receptor Gpr50 readily enter torpor in response to fasting and have now used these mice to conduct a microarray analysis of hypothalamic gene expression changes related to the torpor state. This revealed a strong induction of thioredoxin-interacting protein (Txnip) in the hypothalamus of torpid mice, which was confirmed by quantitative RT-PCR and Western blot analyses. In situ hybridization identified the ependyma lining the third ventricle as the principal site of torpor-related expression of Txnip. To characterize further the relationship between Txnip and torpor, we profiled Txnip expression in mice during prolonged fasting, cold exposure, and 2-deoxyglucose-induced hypometabolism, as well as in naturally occurring torpor bouts in the Siberian hamster. Strikingly, pronounced up-regulation of Txnip expression was only observed in wild-type mice when driven into torpor and during torpor in the Siberian hamster. Increase of Txnip was not limited to the hypothalamus, with exaggerated expression in white adipose tissue, brown adipose tissue, and liver also demonstrated in torpid mice. Given the recent identification of Txnip as a molecular nutrient sensor important in the regulation of energy metabolism, our data suggest that elevated Txnip expression is critical to regulating energy expenditure and fuel use during the extreme hypometabolic state of torpor.

A role for the melatonin-related receptor GPR50 in leptin signaling, adaptive thermogenesis, and torpor.

The ability of mammals to maintain a constant body temperature has proven to be a profound evolutionary advantage, allowing members of this class to thrive in most environments on earth. Intriguingly, some mammals employ bouts of deep hypothermia (torpor) to cope with reduced food supply and harsh climates [1, 2]. During torpor, physiological processes such as respiration, cardiac function, and metabolic rate are severely depressed, yet the neural mechanisms that regulate torpor remain unclear [3]. Hypothalamic responses to energy signals, such as leptin, influence the expression of torpor [4-7]. We show that the orphan receptor GPR50 plays an important role in adaptive thermogenesis and torpor. Unlike wild-type mice, Gpr50(-/-) mice readily enter torpor in response to fasting and 2-deoxyglucose administration. Decreased thermogenesis in Gpr50(-/-) mice is not due to a deficit in brown adipose tissue, the principal site of nonshivering thermogenesis in mice [8]. GPR50 is highly expressed in the hypothalamus of several species, including man [9, 10]. In line with this, altered thermoregulation in Gpr50(-/-) mice is associated with attenuated responses to leptin and a suppression of thyrotropin-releasing hormone. Thus, our findings identify hypothalamic circuits involved in torpor and reveal GPR50 to be a novel component of adaptive thermogenesis in mammals.

GPR50 interacts with TIP60 to modulate glucocorticoid receptor signalling.

GPR50 is an orphan G-protein coupled receptor most closely related to the melatonin receptors. The physiological function of GPR50 remains unclear, although our previous studies implicate the receptor in energy homeostasis. Here, we reveal a role for GPR50 as a signalling partner and modulator of the transcriptional co-activator TIP60. This interaction was identified in a yeast-two-hybrid screen, and confirmed by co-immunoprecipitation and co-localisation of TIP60 and GPR50 in HEK293 cells. Co-expression with TIP60 increased perinuclear localisation of full length GPR50, and resulted in nuclear translocation of the cytoplasmic tail of the receptor, suggesting a functional interaction of the two proteins. We further demonstrate that GPR50 can enhance TIP60-coactiavtion of glucocorticoid receptor (GR) signalling. In line with in vitro results, repression of pituitary Pomc expression, and induction of gluconeogenic genes in liver in response to the GR agonist, dexamethasone was attenuated in Gpr50(-/-) mice. These results identify a novel role for GPR50 in glucocorticoid receptor signalling through interaction with TIP60.