Rev-erbα Expression Research Articles

Pharmacological modulation and genetic deletion of REV-ERBα and REV-ERBβ regulates dendritic cell development

The nuclear receptors REV-ERBα and REV-ERBβ have been demonstrated to play key roles in the regulation of numerous physiological functions, such as metabolism and the circadian rhythm. Recent studies have established the REV-ERBs’ roles in immunity, including macrophage and T cell responses. In contrast, their roles in dendritic cells have not been well defined. Dendritic cells are potent antigen presenting cells, connecting microbial sensing and innate immunity to adaptive immune responses. We demonstrate that both REV-ERBα and REV-ERBβ expression is upregulated during the course of bone marrow derived dendritic cell (BMDC) differentiation. BMDCs from REV-ERBα and REV-ERBβ deficient mice showed enhanced expression of maturation markers like CD86, MHCII, and proinflammatory cytokines. Conversely, treatment of BMDCs with a REV-ERB-specific agonist, SR9009, inhibited the expression of maturation markers and proinflammatory cytokines. Our study suggests the REV-ERBs act as negative regulators of dendritic cell development and activation. These results indicate that pharmacological modulation of REV-ERB activity could be an attractive strategy to modulate DC activation status and for DC-based therapies.

Rev-erbαheterozygosity produces a dose-dependent phenotypic advantage in mice.

Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erbα) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erbα expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erbα ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erbα loss, curiously stimulated these processes. Our investigations revealed that Rev-erbα dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.

Roles of HDAC3-Orchestrated Circadian Clock Gene Oscillations in Diabetic Rats Following Myocardial Ischemia / Reperfusion Injury

Background: Circadian clock has been closely related to the development of diabetes mellitus and cardiovascular disease; the disruption of circadian clock exacerbates myocardial ischemia/reperfusion injury (MI/RI). HDAC3 is a key component of the circadian negative feedback loop by recruitmenting the expression pattern of circadian nuclear receptor Rev-erbα, then to maintain the stability of circadian gene such as BMAL1. In this research, we explored the mechanism of HDAC3-orchestrated Rev-erbα/BMAL1 pathway in increasing MI/RI vulnerability of diabetes, and its relationship with mitophagy. Methods: Streptozocin (STZ) was used to establish type 1 diabetes by intraperitoneal injection. After 8 weeks, the diabetic and non-diabetic rats were exposed to MI/RI by ligating the left anterior descending coronary artery (LDA) for 30 minutes and reperfusion for 120 minutes at four time points of zeitgeber (ZT) 0, 6, 12 and 18. In vitro experiments, primary cardiomyocytes with or without HDAC3 siRNA and Rev-erbα siRNA exposed to hypoxia/reoxygenation (H/R). Findings: Circadian clock gene oscillations were rapidly attenuated in diabetic I/RI hearts, versus sham and/or the non-diabetic I/RI hearts, in accord with circadian mitophagy. By utilizing AAV-HDAC3 to knockdown HDAC3 expression, HDAC3 deficiency significantly attenuated diabetic MI/RI associated with increased autophagy activation. The expression of HDAC3 and Rev-erbα in cardiomyocytes was increased under high glucose condition with decreased BMAL1 expression and autophagy level. After H/R stimulation, the cell injury was obviously increased with upregulated HDAC3 and Rev-erbα expression and decreased BMAL1 level. Moreover, high glucose aggravated H/R injury compared with low glucose by reducing autophagy level, increasing the levels of HDAC3 and Rev-erbα and down-regulating BMAL1 expression. HDAC3 and Rev-erbα siRNA can alleviate high glucose and H/R-induced injury by up-regulating BMAL1 expression and mitophagy levels. Interpretation: These findings suggest that in the MI/RI of diabetic rat, the circadian clocks were rapidly impaired then to inhibit mitophagy; cardiac-targeted manipulation of HDAC3-orchestrated Rev-erbα/BMAL1 pathway may be the mechanism of the decreased toleration to ischemia with a circadian dependent variability. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China 81970722, 81671891 and 81701891. Declaration of Interests: The authors declare that there is no conflict of interests regarding the publication of this paper. Ethics Approval Statement: Experimental protocols were implemented after being reviewed and approved by the Laboratory Animal Welfare & Ethics Committee (IACUC) of Renmin Hospital of Wuhan University. All researchers have obtained the certificate of professional laboratory animal technology in Hubei Province.

Central administration of REV-ERBα agonist promotes opposite responses on energy balance in fasted and fed states.

The REV-ERBα receptor has a recognised role in the regulation of the circadian rhythm system. However, recent evidence suggests that it also contributes to energy balance regulation. Both expression and function of REV-ERBα can be influenced by the energy status of the body. Considering the possibility of the involvement of REV-ERBα in the regulation of energy balance, which is critically regulated by the hypothalamus, and based on the impact of intermittent fasting, the present study evaluated the effects of central administration of REV-ERBα agonist on energy balance in rats exposed to 24hours of fasting or ad lib. feeding conditions. Initially, 24-hour fasted rats received an acute i.c.v. administration of agonist at doses of 1, 5, 10 or 15μgper rat and feed efficiency was evaluated. Because 10μg was a sufficient dose to affect feed efficiency, subsequent experiments used this dose to assess effects of agonist on the following parameters: energy expenditure induced by physical activity and locomotor activity, time spent in physical activity over 24hours, and glucose and insulin tolerance. In fasted rats, the agonist promoted increased food intake and feed efficiency, with a greater body weight gain associated with less time spent in locomotor activity, suggesting a reduction in energy expenditure induced by physical activity. Furthermore, a reduction in glucose tolerance was noted. By contrast, free-fed rats exhibited reduced food intake and feed efficiency with decreased body weight gain along with an increase in locomotor activity and physical activity-dependent energy expenditure. Thus, i.c.v. administration of REV-ERBα agonist regulates energy balance depending on the energy status of the organism; that is, it promotes a positive energy balance in the fasted state and a negative energy balance in the fed state. These results may be useful in understanding the underlying mechanisms of energy balance disorders and intermittent fasting for body weight control.

Decreased expression of Rev-Erb\u03b1 in the epileptic foci of temporal lobe epilepsy and activation of Rev-Erb\u03b1 have anti-inflammatory and neuroprotective effects in the pilocarpine model

BackgroundA hallmark of temporal lobe epilepsy (TLE) is brain inflammation accompanied by neuronal demise. Accumulating evidence demonstrates that Rev-Erbα is involved in regulating neuroinflammation and determining the fate of neurons. Therefore, we studied the expression and cellular distribution of Rev-Erbα in the epileptogenic zone of TLE and the effect of treatment with the Rev-Erbα specific agonist SR9009 in the pilocarpine model.MethodsThe expression pattern of Rev-Erbα was investigated by western blotting, immunohistochemistry, and immunofluorescence labeling in patients with TLE. Next, the effects of SR9009 on neuroinflammation, neuronal apoptosis, and neuronal loss in the mouse hippocampus 7 days after status epilepticus (SE) were assessed by western blotting, immunofluorescence labeling staining, and TUNEL staining.ResultsThe western blotting, immunohistochemistry, and immunofluorescence labeling results revealed that Rev-Erbα was downregulated in the epileptogenic zone of TLE patients and mainly localized in neurons, astrocytes, and presumably microglia. Meanwhile, the expression of Rev-Erbα was decreased in the hippocampus and temporal neocortex of mice treated with pilocarpine in the early post-SE and chronic phases. Interestingly, the expression of Rev-Erbα in the normal hippocampus showed a 24-h rhythm; however, the rhythmicity was disturbed in the early phase after SE, and this disturbance was still present in epileptic animals. Our further findings revealed that treatment with SR9009 inhibited NLRP3 inflammasome activation, inflammatory cytokine (IL-1β, IL-18, IL-6, and TNF-α) production, astrocytosis, microgliosis, and neuronal damage in the hippocampus after SE.ConclusionsTaken together, these results suggested that a decrease in Rev-Erbα in the epileptogenic zone may contribute to the process of TLE and that the activation of Rev-Erbα may have anti-inflammatory and neuroprotective effects.

Chronopharmacological targeting of Rev-erbα by puerarin alleviates hyperhomocysteinemia in mice

Hyperhomocysteinemia is associated with poor health, including cardiovascular and brain diseases. Puerarin, initially isolated from Puerariae radix, has been shown to possess anti-hyperhomocysteinemia effect. However, the mechanism of puerarin action remains unknown. Here, we uncovered that puerarin targeted the circadian clock protein Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. We first identified puerarin as an antagonist of Rev-erbα based on luciferase reporter, Gal4 co-transfection and target gene expression assays. Consistent with an antagonistic effect, puerarin induced mRNA and protein expressions of Bhmt, Cbs and Cth (three enzymes involved in homocysteine catabolism and known targets of Rev-erbα) in Hepa-1c1c7 cells. These induction effects of puerarin were lost in Rev-erbα-deficient cells. Furthermore, puerarin dose-dependently alleviated methionine-induced hyperhomocysteinemia in mice as evidenced by decreased levels of total homocysteine and triglyceride. This was accompanied by increased expressions of Bhmt, Cbs and Cth in the liver. Moreover, puerarin dosed at ZT10 generated stronger pharmacological effects than drug dosed at ZT22 consistent with diurnally rhythmic expression of Rev-erbα (a high expression at ZT10 and a low expression at ZT22). In conclusion, puerarin targets Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. The finding of a circadian gene as drug target encourages chronotherapeutic practices on puerarin and related medications for optimized efficacy.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Circadian pharmacological effects of berberine on chronic colitis in mice: Role of the clock component Rev-erbα

Berberine, initially isolated from Rhizoma Coptidis (Huanglian in Chinese), is a drug used to treat gastrointestinal disorders such as colitis. Here we uncovered a time-varying berberine effect on chronic colitis in mice, and investigated a potential role of the clock protein Rev-erbα in this timing effect. Berberine activity toward Rev-erbα was determined by luciferase reporter, Gal4-cotransfection assay and target gene expression analyses. Chronic colitis was induced by feeding mice with dextran sulfate sodium in drinking water. Colitis severity and pharmacological effects of berberine were assessed by measuring myeloperoxidase and malondialdehyde activities as well as the levels of inflammatory factors (IL-1β, IL-6, IL-18 and Ccl2). Berberine significantly inhibited Bmal1 (−2000/+100 bp)- and Nlrp3 (−1310/+100 bp)-Luc reporter activities, and dose-dependently decreased cellular expressions of both Bmal1 and Nlrp3. Also, it enhanced the transcriptional repressor activity of Rev-erbα in the Gal4 chimeric assay. These data indicated berberine as a Rev-erbα agonist. As expected, berberine attenuated inflammatory responses in BMDMs (bone marrow-derived macrophages) and in colitis mice. However, the anti-inflammatory effects of berberine were lost in BMDMs derived from Rev-erbα-deficient mice. Furthermore, chronic colitis displayed a diurnal rhythmicity in disease severity and its diurnal pattern was in an opposite phase to that of Rev-erbα expression, supporting a direct control of colitis by Rev-erbα. Moreover, berberine effects on chronic colitis were dosing time-dependent. ZT10 dosing generated a better treatment outcome compared to ZT2. This was because colitis was less severe and Rev-erbα expression was much higher at ZT10 than at ZT2. In conclusion, circadian pharmacological effects of berberine on chronic colitis were mainly contributed by diurnal rhythms of both disease severity and Rev-erbα (as a drug target). The findings may have implications for chronotherapeutic practice on colitis or related diseases.

Rev-erb\u03b1 inhibits proliferation by reducing glycolytic flux and pentose phosphate pathway in human gastric cancer cells

Rev-erbα is a nuclear receptor, which regulates circadian rhythm, inflammatory responses and lipid metabolism. We previously showed Rev-erbα reduction in human gastric cancer, which is associated with TMN stages and poor prognosis. We hypothesized that Rev-erbα modulates proliferation via glycolytic flux and the pentose phosphate pathway (PPP) in gastric cancer. Knockdown of Rev-erbα significantly increased proliferation as well as glycolytic flux and the PPP in human gastric cancer cells. These effects were reduced by a Rev-erbα agonist GSK4112 in a dose-dependent manner. Furthermore, Rev-erbα was recruited on the promoters of PFKFB3 and G6PD genes, thereby inhibiting their gene transcription. GSK4112 treatment reduced PFKFB3 and G6PD gene expression, which was not affected by BMAL1 knockdown. Pharmacological inhibition of glycolysis and the PPP using corresponding PFKFB3 and G6PD inhibitors attenuated Rev-erbα knockdown-induced proliferation in gastric cancer cells. GSK4112 treatment was not able to reduce proliferation in SGC-7901 overexpressing both PFKFB3 and G6PD genes. Both PFKFB3 and G6PD were overexpressed in patients with gastric cancer, and positively correlated with the TMN stages. The PPP and glycolysis were enhanced in gastric cancer tissues of patients with low expression of Rev-erbα compared to the patients with high expression of Rev-erbα. In conclusion, Rev-erbα reduction causes gastric cancer progression by augmenting the PPP and glycolysis.

The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.

325-LB: Circadian Clock Nuclear Receptor REV-ERBa Is a Novel Regulator of Beta-Cell Function, Survival, and Autophagy under Diabetogenic Conditions

The circadian clock regulates diverse cellular and molecular rhythms employing CLOCK-BMAL1 transcriptional heterodimer with nuclear receptor REV-ERBα (encoded by gene Nr1d1) playing an important role as a clock repressor through modulation of Bmal1 transcription. Importantly, in addition to its core circadian clock function, recent studies have identified REV-ERBα as a potent transcriptional repressor of autophagy. Therefore, in the current study we set out to address whether impaired beta-cell function and survival associated with exposure to diabetogenic stressors (e.g., glucotoxicity and inflammation) is attributed in part to REV-ERBα-mediated inhibition of autophagy. Exposure of beta-cells (INS-1E cell line) to either glucotoxicity (30 mM glucose) or cytokines (cytomix of IL-1β, TNFα and IFNγ) resulted in robust induction of REV-ERBα expression (1.5-2 fold, p<0.05) and corresponded with impaired autophagy flux characterized by increased protein levels of p62 (also known SQSTM1) (1.5-2 fold, p<0.05). Consistent with these data, exposure of beta-cells to a REV-ERBα agonist (SR9011) was characterized by impaired autophagy (increased p62 levels and aggregated forms, p<0.05), defective glucose-stimulated insulin secretion (70% decrease) and increased beta-cell apoptosis (increased cleaved caspase-3, p<0.01 vs. vehicle). In contrast, REV-ERBα specific antagonist (SR8278) partly protected beta-cells from deleterious effects of glucotoxicity or cytokines-induced inflammation by enhancing autophagy flux and attenuating beta-cell apoptosis (∼30%). Taken together, these data reveal for the first time an underexplored link between the core circadian clock nuclear receptor REV-ERBα, autophagy and beta-cell failure under diabetogenic conditions. These data also suggest a therapeutic potential of modulating REV-ERBα levels in beta-cells to enhance function and survival in diabetes. Disclosure S. Costes: None. D. Laouteouet: None. M.A. Ravier: None. M. Delobel: None. G. Bertrand: None. S. Dalle: None. A. Matveyenko: None.

Nutrient-sensitive transcription factors TFEB and TFE3 couple autophagy and metabolism to the peripheral clock.

Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.

The Absence of Pineal Melatonin Abolishes the Daily Rhythm of Tph1 (Tryptophan Hydroxylase 1), Asmt (Acetylserotonin O-Methyltransferase), and Aanat (Aralkylamine N-Acetyltransferase) mRNA Expressions in Rat Testes.

This study examined the effects of pinealectomy in Wistar rats and melatonin replacement therapy on the daily mRNA expression of melatonin (Tph1, Aanat, Asmt, Mt1, Mt2, and Rorα), and steroidogenic (Star, 17βhsd3, and Lhr) related genes as well as clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, and Cry2) in testes. The testes of control animals express the Tph1, Aanat, and Asmt and Per2 genes with 24-h rhythms in mRNA, reaching the maximal values during the dark phase. Pinealectomy abolished and melatonin treatment restored the 24-h rhythmicity. Daytime differences in mRNA expression were significant for Star, Lhr, Mt1, Mt2, Rorα, Rev-erbα, Bmal1, Cry1, and Cry2 genes in testes of control rats. Conversely, 17βhsd3 and Per1 mRNA expression did not show a daytime difference in testes of control animals. Pinealectomy abolished the peak time of Mt1 and Mt2 mRNA expression, phase shifted the peak time of Star, Rorα, Rev-erbα, Bmal1, and Cry2 mRNA expression, downregulated the 24-h Lhr mRNA expression, and inverted the peak time of Per1, Per2, and Cry1 mRNA expression to the light phase. The melatonin replacement therapy completely restored the control levels of Lhr, Rev-erbα, and Per1 mRNA expression patterns, partially restored the daily control of Star, Mt2, Rorα, Bmal1, Cry1, and Cry2 mRNA expression but did not re-establish the daily control of Mt1 mRNA expression. This suggests that the daily mRNA expression of these genes is probably driven by pineal melatonin and melatonin treatment restores (partially or completely) the daily control of gene expression patterns.

Diurnal Profiles of N-Acylethanolamines in Goldfish Brain and Gastrointestinal Tract: Possible Role of Feeding.

N-acylethanolamines (NAEs) are a family of endogenous lipid signaling molecules that are involved in regulation of energy homeostasis in vertebrates with a putative role on circadian system. The aim of this work was to study the existence of daily fluctuations in components of NAEs system and their possible dependence on food intake. Specifically, we analyzed the content of oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), their precursors (NAPEs), as well as the expression of nape-pld (NAEs synthesis enzyme), faah (NAEs degradation enzyme), and pparα (NAEs receptor) in gastrointestinal and brain tissues of goldfish (Carassius auratus) throughout a 24-h cycle. Daily profiles of bmal1a and rev-erbα expression in gastrointestinal tissues were also quantified because these clock genes are also involved in lipid metabolism, are PPAR-targets in mammals, and could be a link between NAEs and circadian system in fish. Gastrointestinal levels of NAEs exhibited daily fluctuations, with a pronounced and rapid postprandial increase, the increment being likely caused by food intake as it is not present in fasted animals. Such periprandial differences were not found in brain, supporting that NAEs mobilization occurs in a tissue-specific manner and suggesting that these three NAEs could be acting as peripheral satiety signals. The abundance of pparα mRNA displayed a daily rhythm in the intestine and the liver, suggesting a possible rhythmicity in the NAEs functionality. The increment of pparα expression during the rest phase can be related with its role stimulating lipid catabolism to obtain energy during the fasting state of the animals. In addition, the clock genes bmal1a and rev-erbα also showed daily rhythms, with a bmal1a increment after feeding, supporting its role as a lipogenic factor. In summary, our data show the existence of all components of NAEs system in fish (OEA, PEA, SEA, precursors, synthesis and degradation enzymes, and the receptor PPARα), supporting the involvement of NAEs as peripheral satiety signals.

REV-ERB mediated regulation of dendritic cell maturation and function

Abstract The REV-ERBs (REV-ERBα and REV-ERBβ) are members of the nuclear receptor superfamily of ligand-regulated transcription factors and owing to their ability to recruit corepressor, act as transcriptional repressors. The REV-ERBs have well-established roles in regulating various physiological functions, including metabolism and the circadian rhythm. However, little is known about their roles in the immune system. While some evidence exists suggesting the REV-ERBs, particularly REV-ERBα, regulates macrophage inflammatory responses, the REV-ERB’s role in regulating other cells of myeloid origin remains largely unknown. Dendritic cells (DCs), like macrophages, are of myeloid origin and serve as the master regulator of immune responses by connecting microbial sensing and innate immunity to adaptive immune responses. Given the central role of DCs in regulating immune responses, we investigated if the REV-ERBs regulate DC maturation and function. We found that both REV-ERBα and REV-ERBβ expression is upregulated during the course of bone marrow derived dendritic cell (BMDCs) differentiation. BMDCs from REV-ERBα and REV-ERBβ deficient mice showed enhanced expression of maturation markers like CD86, MHCII, CD83 and proinflammatory cytokines. In contrast, treatment of BMDCs with a REV-ERB-specific agonist, SR9009, inhibited the expression of these maturation markers and proinflammatory cytokines. Our study suggests the REV-ERBs act as negative regulators of dendritic cell maturation and function thus pharmacological modulation of REV-ERB activity could be an attractive strategy in treating inflammatory diseases.

CDK9 modulates circadian clock by attenuating REV-ERBα activity

Circadian clock and cell cycle are vital cellular programs acting in a timely-regulated, cyclic manner. The two cellular oscillators are coupled in various ways to facilitate biological processes. Here we report CDK9, a kinase belongs to the CDK family in regulating cell cycle and RNA Pol II activity, can serve as a modulator for circadian clock. We identified CDK inhibitor LY2857785 potently blocked PER2:LUC expression in MEFs from a screen of 17 commonly-used CDK inhibitors. We further analyzed the possible targets of LY2857785 by siRNA approach, and confirmed CDK9 as the main effector. LY2857785 treatment, as well as Cdk9 knock-down, led to lowered expression of Bmal1 in accordance with elevated expression of Rev-Erbα. CDK9 associated with REV-ERBα thus attenuated REV-ERBα binding to the RORE for Bmal1 suppression. To conform the circadian-modulating activity of CDK9 in vivo, we knocked down CDK9 in mice at the anterior hypothalamus covering the central oscillator SCN, and found the respiratory exchange ratio, daily activity and circadian period were altered in the Cdk9-knockdown mice. Together, our finding designated CDK9 as a novel modulator in circadian clock. CDK9 may serve as a vital basis to understand circadian- and cell cycle-misregulated ailments such as cancer.

Disruption of central and peripheral circadian clocks in police officers working at night.

Working atypical schedules leads to temporal misalignments between a worker's rest-activity cycle and their endogenous circadian system. Several studies have reported disturbed centrally controlled rhythms, but little is known on shift workers' peripheral clocks. Here, we assessed central clock markers, urinary 6-sulfatoxymelatonin and salivary cortisol, and clock gene expression in 2 peripheral clocks, oral mucosa cells and peripheral blood mononuclear cells (PBMCs), in 11 police officers. Before working 7 consecutive nights, officers' centrally controlled rhythms were aligned to a day-oriented schedule. These rhythms were partially realigned to the shifted schedule and dampened after a week working nights. For peripheral clocks at baseline, Period (PER)1-3 and nuclear receptor subfamily 1, group D, member 1 (REV-ERBα) in oral mucosa cells had a significant mRNA peak in the afternoon, whereas in PBMCs, higher PER1-3 expression was observed at 10:00 compared with 19:30. After a week working nights, PER1-3 and REV-ERBα expression in oral mucosa cells lost rhythmicity, and in PBMCs, the morning/evening difference observed at baseline was lost. To our knowledge, this is the first study to demonstrate the disruption of several peripheral clocks in real shift workers. Molecular circadian disturbances are believed to have important clinical implications for the occurrence of shift work-associated medical disorders.-Koshy, A., Cuesta, M., Boudreau, P., Cermakian, N., Boivin, D. B. Disruption of central and peripheral circadian clocks in police officers working at night.

Glucocorticoid receptor suppresses gene expression of Rev-erbα (Nr1d1) through interaction with the CLOCK complex.

Glucocorticoids have various medical uses but are accompanied by side effects. The glucocorticoid receptor (GR) has been reported to regulate the clock genes, but the underlying mechanisms are incompletely understood. In this study, we focused on the suppressive effect of the GR on the expression of Rev-erbα (Nr1d1), an important component of the clock regulatory circuits. Here we show that the GR suppresses Rev-erbα expression via the formation of a complex with CLOCK and BMAL1, which binds to the E-boxes in the Nr1d1 promoter. In this GR-CLOCK-BMAL1 complex, the GR does not directly bind to DNA, which is referred to as tethering. These findings provide new insights into the role of the GR in the control of circadian rhythm.

Bisphenol A Alters Bmal1, Per2, and Rev-Erba mRNA and Requires Bmal1 to Increase Neuropeptide Y Expression in Hypothalamic Neurons.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is considered an obesogen. However, its role in the hypothalamic control of energy balance remains largely unexplored. Because disruption of the circadian clock is tightly associated with metabolic consequences, we explored how BPA affects the components of the molecular circadian clock in the feeding-related neurons of the hypothalamus. In immortalized POMC and NPY/AgRP-expressing hypothalamic cell lines and primary culture, we describe how BPA significantly alters mRNA expression of circadian clock genes Bmal1,Per2, and Rev-Erbα. Furthermore, we use newly generated Bmal1-knockout (KO) hypothalamic cell lines to link the BPA-induced neuropeptide dysregulation to the molecular clock. Specifically, BPA increased Npy, Agrp, and Pomc mRNA expression in wild type hypothalamic cells, whereas the increase in Npy, but not Agrp or Pomc, was abolished in cell lines lacking BMAL1. In line with this increase, BPA led to increased BMAL1 binding to the Npy promotor, potentially increasing Npy transcription. In conclusion, we show that BPA-mediated dysregulation of the circadian molecular clock is linked to the deleterious effects of BPA on neuropeptide expression. Furthermore, we describe hypothalamic Bmal1-KO cell lines to study the role of BMAL1 in hypothalamic responses to metabolic, hormonal, and environmental factors.

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex

The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα-interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.