Pharmacological inhibition of cryptochrome and REV-ERB promotes DNA repair and cell cycle arrest in cisplatin-treated human cells.

Nucleotide excision repair (NER) and cell cycle checkpoints impact the ability of the anti-cancer drug cisplatin to inhibit cell proliferation and induce cell death. Genetic studies have shown that both NER and cell cycle progression are impacted by the circadian clock, which has emerged as a novel pharmacological target for the treatment of various disease states. In this study, cultured human cell lines were treated with combinations of cisplatin and the circadian clock modulating compounds KS15 and SR8278, which enhance circadian clock transcriptional output by inhibiting the activities of the cryptochrome and REV-ERB proteins, respectively. Treatment of cells with KS15 and SR8278 protected cells against the anti-proliferative effects of cisplatin and increased the expression of NER factor XPA and cell cycle regulators Wee1 and p21 at the mRNA and protein level. Correlated with these molecular changes, KS15 and SR8278 treatment resulted in fewer unrepaired cisplatin-DNA adducts in genomic DNA and a higher fraction of cells in the G1 phase of the cell cycle. Thus, the use of pharmacological agents targeting the circadian clock could be a novel approach to modulate the responses of normal and cancer cells to cisplatin chemotherapy regimens.

Deficiency of intestinal Bmal1 prevents obesity induced by high-fat feeding.

The role of intestine clock in energy homeostasis remains elusive. Here we show that mice with Bmal1 specifically deleted in the intestine (Bmal1iKO mice) have a normal phenotype on a chow diet. However, on a high-fat diet (HFD), Bmal1iKO mice are protected against development of obesity and related abnormalities such as hyperlipidemia and fatty livers. These metabolic phenotypes are attributed to impaired lipid resynthesis in the intestine and reduced fat secretion. Consistently, wild-type mice fed a HFD during nighttime (with a lower BMAL1 expression) show alleviated obesity compared to mice fed ad libitum. Mechanistic studies uncover that BMAL1 transactivates the Dgat2 gene (encoding the triacylglycerol synthesis enzyme DGAT2) via direct binding to an E-box in the promoter, thereby promoting dietary fat absorption. Supporting these findings, intestinal deficiency of Rev-erbα, a known BMAL1 repressor, enhances dietary fat absorption and exacerbates HFD-induced obesity and comorbidities. Moreover, small-molecule targeting of REV-ERBα/BMAL1 by SR9009 ameliorates HFD-induced obesity in mice. Altogether, intestine clock functions as an accelerator in dietary fat absorption and targeting intestinal BMAL1 may be a promising approach for management of metabolic diseases induced by excess fat intake.

The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.

Inhibition of Rev-erbα ameliorates muscular dystrophy.

Duchene muscular dystrophy leads to progressive muscle structural and functional decline due to chronic degenerative-regenerative cycles. Enhancing the regenerative capacity of dystrophic muscle provides potential therapeutic options. We previously demonstrated that the circadian clock repressor Rev-erbα inhibited myogenesis and Rev-erbα ablation enhanced muscle regeneration. Here we show that Rev-erbα deficiency in the dystrophin-deficient mdx mice promotes regenerative myogenic response to ameliorate muscle damage. Loss of Rev-erbα in mdx mice improved dystrophic pathology and muscle wasting. Rev-erbα-deficient dystrophic muscle exhibit augmented myogenic response, enhanced neo-myofiber formation and attenuated inflammatory response. In mdx myoblasts devoid of Rev-erbα, myogenic differentiation was augmented together with up-regulation of Wnt signaling and proliferative pathways, suggesting that loss of Rev-erbα inhibition of these processes contributed to the improvement in regenerative myogenesis. Collectively, our findings revealed that the loss of Rev-erbα function protects dystrophic muscle from injury by promoting myogenic repair, and inhibition of its activity may have therapeutic utilities for muscular dystrophy.

The Effect of Rev-erbα Agonist SR9011 on the Immune Response and Cell Metabolism of Microglia.

Microglia are the immune cells of the brain. Hyperactivation of microglia contributes to the pathology of metabolic and neuroinflammatory diseases. Evidence has emerged that links the circadian clock, cellular metabolism, and immune activity in microglia. Rev-erb nuclear receptors are known for their regulatory role in both the molecular clock and cell metabolism, and have recently been found to play an important role in neuroinflammation. The Rev-erbα agonist SR9011 disrupts circadian rhythm by altering intracellular clock machinery. However, the exact role of Rev-erbα in microglial immunometabolism remains to be elucidated. In the current study, we explored whether SR9011 also had such a detrimental impact on microglial immunometabolic functions. Primary microglia were isolated from 1-3 days old Sprague-Dawley rat pups. The expression of clock genes, cytokines and metabolic genes was evaluated using RT-PCR and rhythmic expression was analyzed. Phagocytic activity was determined by the uptake capacity of fluorescent microspheres. Mitochondria function was evaluated by measuring oxygen consumption rate and extracellular acidification rate. We found that key cytokines and metabolic genes are rhythmically expressed in microglia. SR9011 disturbed rhythmic expression of clock genes in microglia. Pro-inflammatory cytokine expression was attenuated by SR9011 during an immune challenge by TNFα, while expression of the anti-inflammatory cytokine Il10 was stimulated. Moreover, SR9011 decreased phagocytic activity, mitochondrial respiration, ATP production, and metabolic gene expression. Our study highlights the link between the intrinsic clock and immunometabolism of microglia. We show that Rev-erbα is implicated in both metabolic homeostasis and the inflammatory responses in microglia, which has important implications for the treatment of metabolic and neuroinflammatory diseases.

The transcriptional repressor REV-ERB as a novel target for disease.

REV-ERB is a member of the nuclear receptor superfamily of transcription factors involved in the regulation of many physiological processes, from circadian rhythm, to immune function and metabolism. Accordingly, REV-ERB has been considered as a promising, but difficult drug target for the treatment of numerous diseases. Here, we concisely review current understanding of the function of REV-ERB, modulation by endogenous factors and synthetic ligands, and the involvement of REV-ERB in select human diseases. Particular focus is placed on the medicinal chemistry of synthetic REV-ERB ligands, which demonstrates the need for higher quality ligands to aid in robust validation of this exciting target.

Targeting REV-ERBα for therapeutic purposes: promises and challenges.

REV-ERBα (NR1D1) is a circadian clock component that functions as a transcriptional repressor. Due to its role in direct modulation of metabolic genes, REV-ERBα is regarded as an integrator of cell metabolism with circadian clock. Accordingly, REV-ERBα is first proposed as a drug target for treating sleep disorders and metabolic syndromes (e.g., dyslipidaemia, hyperglycaemia and obesity). Recent years of studies uncover a rather broad role of REV-ERBα in pathological conditions including local inflammatory diseases, heart failure and cancers. Moreover, REV-ERBα is involved in regulation of circadian drug metabolism that has implications in chronopharmacology. In the meantime, recent years have witnessed discovery of an array of new REV-ERBα ligands most of which have pharmacological activities in vivo. In this article, we review the regulatory role of REV-ERBα in various types of diseases and discuss the underlying mechanisms. We also describe the newly discovered ligands and the old ones together with their targeting potential. Despite well-established pharmacological effects of REV-ERBα ligands in animals (preclinical studies), no progress has been made regarding their translation to clinical trials. This implies certain challenges associated with drug development of REV-ERBα ligands. In particular, we discuss the potential challenges related to drug safety (or adverse effects) and bioavailability. For new drug development, it is advocated that REV-ERBα should be targeted to treat local diseases and a targeting drug should be locally distributed, avoiding the adverse effects on other tissues.

Inhibition of REV-ERBs stimulates microglial amyloid-beta clearance and reduces amyloid plaque deposition in the 5XFAD mouse model of Alzheimer's disease.

A promising new therapeutic target for the treatment of Alzheimer's disease (AD) is the circadian system. Although patients with AD are known to have abnormal circadian rhythms and suffer sleep disturbances, the role of the molecular clock in regulating amyloid-beta (Aß) pathology is still poorly understood. Here, we explored how the circadian repressors REV-ERBα and ß affected Aß clearance in mouse microglia. We discovered that, at Circadian time 4 (CT4), microglia expressed higher levels of the master clock protein BMAL1 and more rapidly phagocytosed fibrillary Aß1-42 (fAß1-42 ) than at CT12. BMAL1 directly drives transcription of REV-ERB proteins, which are implicated in microglial activation. Interestingly, pharmacological inhibition of REV-ERBs with the small molecule antagonist SR8278 or genetic knockdown of REV-ERBs-accelerated microglial uptake of fAß1-42 and increased transcription of BMAL1. SR8278 also promoted microglia polarization toward a phagocytic M2-like phenotype with increased P2Y12 receptor expression. Finally, constitutive deletion of Rev-erbα in the 5XFAD model of AD decreased amyloid plaque number and size and prevented plaque-associated increases in disease-associated microglia markers including TREM2, CD45, and Clec7a. Altogether, our work suggests a novel strategy for controlling Aß clearance and neuroinflammation by targeting REV-ERBs and provides new insights into the role of REV-ERBs in AD.

Meiotic gatekeeper STRA8 suppresses autophagy by repressing Nr1d1 expression during spermatogenesis in mice.

The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation.

Rev-erbα can regulate the NF-κB/NALP3 pathway to modulate lipopolysaccharide-induced acute lung injury and inflammation.

Progressive lung injury and pulmonary inflammation can be induced by an intraperitoneal injection of lipopolysaccharide (LPS). Interleukin-1ß (IL-1ß) is a key pro-inflammatory cytokine that can further exaggerate inflammation, which is cleaved and activated by the NALP3 inflammasome. Although the nuclear receptor Rev-erbα attenuates the level of LPS-induced pulmonary inflammation, the mechanism remains unclear. In this study, we investigated the influence of LPS-induced production of IL-1ß and Rev-erbα on the development of lung inflammation. Herein, we demonstrate that Rev-erbα reduces IL-1ß production and lung injury following an intraperitoneal injection of LPS, which is dependent on the NF-κB/NALP3 pathway. Thus, Rev-erbα is able to decrease the extent of acute lung injury by regulating IL-1ß production. This mechanism may represent a potential novel therapeutic approach for lung injury.

Reverse Erythroblastosis Virus α Antagonism Promotes Homocysteine Catabolism and Ammonia Clearance.

Metabolic homeostasis of amino acids is essential for human health. Here, we aimed to investigate a potential role for the clock component reverse erythroblastosis virus α (Rev-erbα) in circadian regulation of amino acid metabolism. RNA-seq with Rev-erbα-/- mice showed expression changes in genes involved in amino acid metabolism, particularly, the urea cycle and methionine metabolism. Rev-erbα ablation increased hepatic mRNA, protein, and enzymatic activity of betaine homocysteine methyltransferase (Bhmt), cystathionine ß-synthase (Cbs), and cystathionine γ-lyase (Cth) and decreased the levels of plasma and liver homocysteine in mice. Cell-based assays confirmed negative regulation of these three genes by Rev-erbα. Combined luciferase reporter, mobility-shift, and chromatin immunoprecipitation assays identified Rev-erbα as a transcriptional repressor of Bhmt, Cbs, and Cth. Rev-erbα ablation or antagonism alleviated chemical-induced hyperhomocysteinemia in mice. This was accompanied by elevated expressions of Bhmt, Cbs, and Cth. Moreover, Rev-erbα ablation or antagonism promoted urea production and ammonia clearance. Of urea cycle-related genes, arginase 1 (Arg1), ornithine transcarbamylase (Otc), and carbamoyl-phosphate synthase 1 (Cps1) expressions were up-regulated in Rev-erbα-/- mice. Negative regulation of these urea cycle genes by Rev-erbα was validated using cell-based experiments. Mechanistic studies revealed that Rev-erbα inhibited CCAAT-enhancer-binding protein α transactivation to repress the transcription of Arg1, Cps1, and Otc. Conclusion: Rev-erbα antagonism alleviates hyperhomocysteinemia and promotes ammonia clearance. Targeting Rev-erbα represents an approach for the management of homocysteine- and ammonia-related diseases.

Distinct roles for REV-ERBα and REV-ERBß in oxidative capacity and mitochondrial biogenesis in skeletal muscle.

The nuclear receptors REV-ERBα and REV-ERBß have been demonstrated to be core members of the circadian clock and participate in the regulation of a diverse set of metabolic functions. Due to their overlapping tissue expression patterns and gene expression profiles, REV-ERBß is thought to be redundant to REV-ERBα. Recent work has highlighted REV-ERBα's role in the regulation of skeletal muscle oxidative capacity and mitochondrial biogenesis. Considering the similarity between the REV-ERBs and the hypothesized overlap in function, we sought to determine whether REV-ERBß-deficiency presented with a similar skeletal muscle phenotype as REV-ERBα-deficiency. Ectopic overexpression in C2C12 cells demonstrated that REV-ERBß drives mitochondrial biogenesis and the expression of genes involved in fatty acid oxidation. Intriguingly, knock down of REV-ERBß in C2C12 cultures also resulted in mitochondrial biogenesis and increased expression of genes involved in fatty acid ß-oxidation. To determine whether these effects occurred in vivo, we examined REV-ERBß-deficient mice and observed a similar increase in expression of genes involved in mitochondrial biogenesis and fatty acid ß-oxidation. Consistent with these results, REV-ERBß-deficient mice exhibited an altered metabolic phenotype compared to wild-type littermate controls when measured by indirect calorimetry. This likely compensated for the increased food consumption that occurred, possibly aiding in the maintenance of their weight over time. Since feeding behaviors are a direct circadian output, this study suggests that REV-ERBß may have more subtle effects on circadian behaviors than originally identified. Furthermore, these data implicate REV-ERBß in the control of skeletal muscle metabolism and energy expenditure and suggest that development of REV-ERBα versus REV-ERBß selective ligands may have therapeutic utility in the treatment of metabolic syndrome.

Daytime variation of perioperative myocardial injury in cardiac surgery and its prevention by Rev-Erbα antagonism: a single-centre propensity-matched cohort study and a randomised study.

BACKGROUND: On-pump cardiac surgery provokes a predictable perioperative myocardial ischaemia-reperfusion injury which is associated with poor clinical outcomes. We determined the occurrence of time-of-the-day variation in perioperative myocardial injury in patients undergoing aortic valve replacement and its molecular mechanisms. METHODS: We studied the incidence of major adverse cardiac events in a prospective observational single-centre cohort study of patients with severe aortic stenosis and preserved left ventricular ejection fraction (>50%) who were referred to our cardiovascular surgery department at Lille University Hospital (Lille, France) for aortic valve replacement and underwent surgery in the morning or afternoon. Patients were matched into pairs by propensity score. We also did a randomised study, in which we evaluated perioperative myocardial injury and myocardial samples of patients randomly assigned (1:1) via permuted block randomisation (block size of eight) to undergo isolated aortic valve replacement surgery either in the morning or afternoon. We also evaluated human and rodent myocardium in ex-vivo hypoxia-reoxygenation models and did a transcriptomic analysis in myocardial samples from the randomised patients to identify the signalling pathway(s) involved. The primary objective of the study was to assess whether myocardial tolerance of ischaemia-reperfusion differed depending on the timing of aortic valve replacement surgery (morning vs afternoon), as measured by the occurrence of major adverse cardiovascular events (cardiovascular death, myocardial infarction, and admission to hospital for acute heart failure). The randomised study is registered with ClinicalTrials.gov, number NCT02812901. FINDINGS: In the cohort study (n=596 patients in matched pairs who underwent either morning surgery [n=298] or afternoon surgery [n=298]), during the 500 days following aortic valve replacement, the incidence of major adverse cardiac events was lower in the afternoon surgery group than in the morning group: hazard ratio 0·50 (95% CI 0·32-0·77; p=0·0021). In the randomised study, 88 patients were randomly assigned to undergo surgery in the morning (n=44) or afternoon (n=44); perioperative myocardial injury assessed with the geometric mean of perioperative cardiac troponin T release was significantly lower in the afternoon group than in the morning group (estimated ratio of geometric means for afternoon to morning of 0·79 [95% CI 0·68-0·93; p=0·0045]). Ex-vivo analysis of human myocardium revealed an intrinsic morning-afternoon variation in hypoxia-reoxygenation tolerance, concomitant with transcriptional alterations in circadian gene expression with the nuclear receptor Rev-Erbα being highest in the morning. In a mouse Langendorff model of hypoxia-reoxygenation myocardial injury, Rev-Erbα gene deletion or antagonist treatment reduced injury at the time of sleep-to-wake transition, through an increase in the expression of the ischaemia-reperfusion injury modulator CDKN1a/p21. INTERPRETATION: Perioperative myocardial injury is transcriptionally orchestrated by the circadian clock in patients undergoing aortic valve replacement, and Rev-Erbα antagonism seems to be a pharmacological strategy for cardioprotection. Afternoon surgery might provide perioperative myocardial protection and lead to improved patient outcomes compared with morning surgery. FUNDING: Fondation de France, Fédération Française de Cardiologie, EU-FP7-Eurhythdia, Agence Nationale pour la Recherche ANR-10-LABX-46, and CPER-Centre Transdisciplinaire de Recherche sur la Longévité.

Pharmacological inhibition of REV-ERB stimulates differentiation, inhibits turnover and reduces fibrosis in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked disorder that is fatal. DMD patients lack the expression of the structural protein dystrophin caused by mutations within the DMD gene. The absence of functional dystrophin protein results in excessive damage from normal muscle use due to the compromised structural integrity of the dystrophin associated glycoprotein complex. As a result, DMD patients exhibit ongoing cycles of muscle destruction and regeneration that promote inflammation, fibrosis, mitochondrial dysfunction, satellite cell (SC) exhaustion and loss of skeletal and cardiac muscle function. The nuclear receptor REV-ERB suppresses myoblast differentiation and recently we have demonstrated that the REV-ERB antagonist, SR8278, stimulates muscle regeneration after acute injury. Therefore, we decided to explore whether the REV-ERB antagonist SR8278 could slow the progression of muscular dystrophy. In mdx mice SR8278 increased lean mass and muscle function, and decreased muscle fibrosis and muscle protein degradation. Interestingly, we also found that SR8278 increased the SC pool through stimulation of Notch and Wnt signaling. These results suggest that REV-ERB is a potent target for the treatment of DMD.

Protein malnutrition after weaning disrupts peripheral clock and daily insulin secretion in mice.

Changes in nutritional state may alter circadian rhythms through alterations in expression of clock genes. Protein deficiency has a profound effect on body metabolism, but the effect of this nutrient restriction after weaning on biological clock has not been explored. Thus, this study aims to investigate whether the protein restriction affects the daily oscillation in the behavior and metabolic rhythms, as well as expression of clock genes in peripheral tissues. Male C57BL/6 J mice, after weaning, were fed a normal-protein (NP) diet or a low-protein (LP) diet for 8 weeks. Mice fed an LP diet did not show difference in locomotor activity and energy expenditure, but the food intake was increased, with parallel increased expression of the orexigenic neuropeptide Npy and disruption of the anorexigenic Pomc oscillatory pattern in the hypothalamus. LP mice showed disruption in the daily rhythmic patterns of plasma glucose, triglycerides and insulin. Also, the rhythmic expression of clock genes in peripheral tissues and pancreatic islets was altered in LP mice. In pancreatic islets, the disruption of clock genes was followed by impairment of daily glucose-stimulated insulin secretion and the expression of genes involved in exocytosis. Pharmacological activation of REV-ERBα could not restore the insulin secretion in LP mice. The present study demonstrates that protein restriction, leading to development of malnutrition, alters the peripheral clock and metabolic outputs, suggesting that this nutrient provides important entraining cues to regulate the daily fluctuation of biological clock.

MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells.

The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.

Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells.

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes (Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2-O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E2 production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE2 production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

REV-ERBα inhibits the PTGS2 expression in bovine uterus endometrium stromal and epithelial cells exposed to ovarian steroids.

The nuclear receptor REV-ERBα (encoded by NR1D1) has a critical role in metabolism and physiology as well as circadian rhythm. Here, we investigated the possible contribution of clock genes including NR1D1 to the secretion of prostaglandin F2α (PGF2α) from bovine uterine stromal (USCs) and epithelial cells (UECs) by modulating the expression of PTGS2. The circadian oscillation of clock genes in the cells was weak compared with that reported in rodents, but the expression of BMAL1, PER1, and NR1D1 was changed temporally by treatment with ovarian steroids. Significant expression of clock genes including NR1D1 was detected in USCs exposed to progesterone. NR1D1 was also significantly expressed in UECs exposed to estradiol. The expression of PTGS2 was suppressed in USCs exposed to progesterone, while the expression was initially suppressed in UECs exposed to estradiol and then increased after long-term exposure to estradiol. BMAL1 knockdown with specific siRNA caused a significant decrease in the transcript levels of NR1D1 and PTGS2 in USCs, but not in UECs. The production of PGF2α also decreased in USCs after BMAL1 knockdown, while its level did not significantly change in UECs. The transcript level of PTGS2 was increased by treatment with the antagonist of REV-ERBα in both cell types, but the agonist was ineffective. In these two cell types treated with the agonist or antagonist, the PGF2α production coincided well with the PTGS2 expression. Collectively, these results indicate that REV-ERBα plays an inhibitory role in the expression of PTGS2 in both bovine USCs and UECs treated with ovarian steroids.

Structure of REV-ERBß ligand-binding domain bound to a porphyrin antagonist.

REV-ERBα and REV-ERBß are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERBß can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERBß with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERBß indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERBß crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.