Rev-erbα Expression Research Articles

Bmal1- and Per2-mediated regulation of the osteogenic differentiation and proliferation of mouse bone marrow mesenchymal stem cells by modulating the Wnt/β-catenin pathway.

Bmal1 and Per2 are the core components of the circadian clock genes (CCGs). Bmal1-/- mice exhibit premature aging, as indicated by hypotrichosis and osteoporosis, with a loss of proliferation ability. The same occurs in Per2-/- mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. Thus, our study aimed to explore the effects and specific mechanism. Lentiviral and adenoviral vectors were constructed to silence or overexpress Bmal1 or Per2 and MTT, flow cytometry, RT-qPCR, WB, immunohistochemistry, alizarin red staining and ChIP-Seq analyses were applied to identify the possible mechanism. The successful knockdown and overexpression of Bmal1/Per2 were detected by fluorescence microcopy. Flow cytometry found out that Bmal1 or Per2 knockdown resulted in G1-phase cell cycle arrest. RT-qPCR showed the different expression levels of Wnt-3a, c-myc1 and axin2 in the Wnt/β-catenin signaling pathway as well as the gene expression change of Rorα and Rev-erbα. Meanwhile, related proteins such as β-catenin, TCF-1, and P-GSK-3β were detected. ALP activity and the amount of mineral nodules were compared. ChIP-Seq results showed the possible mechanism. Bmal1 and Per2, as primary canonical clock genes, showed synergistic effects on the proliferation and differentiation of BMSCs. They would inhibit the Wnt/β-catenin signaling pathway by downregulating Rorα expression or upregulating Rev-erbα expression, both of which were also key elements of CCGs. And this may be the mechanism by which they negatively regulate the osteogenic differentiation of BMSCs. Bmal1 and Per2 show synergistic effects in the proliferation of BMSCs. In addition, they play a synergistic role in negatively regulating the osteogenic differentiation ability of BMSCs. Bmal1 and Per2 may regulate the aging of BMSCs by altering cell proliferation and osteogenic differentiation through Rorα and Rev-erbα to affect Wnt/β-catenin pathway.

Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice.

Regular environmental light–dark (LD) cycle-regulated period circadian clock 2 (Per2) gene expression is essential for circadian oscillation, nutrient metabolism, and intestinal microbiota balance. Herein, we combined environmental LD cycles with Per2 gene knockout to investigate how LD cycles mediate Per2 expression to regulate colonic and cecal inflammatory and barrier functions, microbiome, and short-chain fatty acids (SCFAs) in the circulation. Mice were divided into knockout (KO) and wild type (CON) under normal light–dark cycle (NLD) and short-light (SL) cycle for 2 weeks after 4 weeks of adaptation. The concentrations of SCFAs in the serum and large intestine, the colonic and cecal epithelial circadian rhythm, SCFAs transporter, inflammatory and barrier-related genes, and Illumina 16S rRNA sequencing were measured after euthanasia during 10:00–12:00. KO decreased the feeding frequency at 0:00–2:00 but increased at 12:00–14:00 both under NLD and SL. KO upregulated the expression of Per1 and Rev-erbα in the colon and cecum, while it downregulated Clock and Bmal1. In terms of inflammatory and barrier functions, KO increased the expression of Tnf-α, Tlr2, and Nf-κb p65 in the colon and cecum, while it decreased Claudin and Occludin-1. KO decreased the concentrations of total SCFAs and acetate in the colon and cecum, but it increased butyrate, while it had no impact on SCFAs in the serum. KO increased the SCFAs transporter because of the upregulation of Nhe1, Nhe3, and Mct4. Sequencing data revealed that KO improved bacteria α-diversity and increased Lachnospiraceae and Ruminococcaceae abundance, while it downregulated Erysipelatoclostridium, Prevotellaceae UCG_001, Olsenella, and Christensenellaceae R-7 under NLD in KO mice. Most of the differential bacterial genus were enriched in amino acid and carbohydrate metabolism pathways. Overall, Per2 knockout altered circadian oscillation in the large intestine, KO improved intestinal microbiota diversity, the increase in Clostridiales abundance led to the reduction in SCFAs in the circulation, concentrations of total SCFAs and acetate decreased, while butyrate increased and SCFAs transport was enhanced. These alterations may potentially lead to inflammation of the large intestine. Short-light treatment had minor impact on intestinal microbiome and metabolism.

Downregulation of REV-ERBα is associated with the progression of lung adenocarcinoma.

BackgroundThe nuclear receptor REV-ERBα (nuclear receptor subfamily 1, Group D member 1, NR1D1) is one of the essential components of the circadian clock which modulates cell proliferation, glucose metabolism, inflammation, and many other biological processes. Modulation of these processes are also relevant to cancer development. Previous studies have suggested that activation of REV-ERBα correlates with cancer cell senescence and death, but how REV-ERBα play roles in tumor progression require further elucidation.MethodsWe investigated the expression of REV-ERBα in clinical samples by immunohistochemistry (IHC). REV-ERBα is downregulated by shorth hairpin RNA (shRNA). The gene expression level of each group was detected by Western blot analysis. The effects of REV-ERBα downregulation on apoptosis and cell cycles was assessed by flow cytometry assay. A549 cell growth curve under different treatments measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cell invasion ability under different treatments was measured by Transwell assay. Immunostaining analysis was also used for evaluating the effects of downregulation of REV-ERBα on nuclear factor-κB (NF-κB).ResultsCompared to 81.8% (54/66) of samples exhibiting a lower expression level of REV-ERBα in cancer tissue than in paired normal tissue, only 18.2% (12/66) were higher or equally expressed in lung cancer tissue. Furthermore, downregulation of REV-ERBα by RNA interference can significantly enhance the transcription of nuclear factor-κB (NF-κB), while the expression of p53 remained the same. Downregulation of REV-ERBα was also shown to stimulate the invasion and promote the proliferation of lung adenocarcinoma cell line A549.ConclusionsOur findings suggest that tumorigenesis and progression of lung carcinoma is relevant to downregulation or inhibition of REV-ERBα. This pathophysiological process also correlates with regulation of the NF-κB signaling pathway, indicating that REV-ERBα is a potential target of lung cancer therapy.

Bmal1 regulates production of larger lipoproteins by modulating cAMP-responsive element-binding protein H and apolipoprotein AIV.

High plasma lipid/lipoprotein levels are risk factors for various metabolic diseases. We previously showed that circadian rhythms regulate plasma lipids and deregulation of these rhythms causes hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1)-deficient mice maintained on a chow or Western diet developed hyperlipidemia, denoted by the presence of higher amounts of triglyceride-rich and apolipoprotein AIV (ApoAIV)-rich larger chylomicron and VLDL due to overproduction. Bmal1 deficiency decreased small heterodimer partner (Shp) and increased microsomal triglyceride transfer protein (MTP), a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates cAMP-responsive element-binding protein H (Crebh) to modulate ApoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh-deficient and ApoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on ApoAIV expression and lipoprotein size. These studies indicate that regulation of ApoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and nuclear receptor subfamily 1 group D member 1 (Nr1D1, Rev-erbα) interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and ApoAIV, through two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia and that avoiding disruptions in circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.

REV-ERBs negatively regulate mineralization of the cementoblasts

ObjectiveThe role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. MethodsExpression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. ResultsStrong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. ConclusionsREV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Period 2 Regulates CYP2B10 Expression and Activity in Mouse Liver.

CYP2B10 is responsible for metabolism and detoxification of many clinical drugs. Here, we aimed to investigate a potential role of Period 2 (PER2) in regulating expression of hepatic CYP2B10. Regulatory effects of PER2 on hepatic expression of CYP2B10 and other enzymes were determined using Per2-deficient mice with exons 4-6 deleted (named Per2 Del4-6 mice). In vitro and in vivo metabolic activities of CYP2B10 were probed using cyclophosphamide (CPA) as a specific substrate. Regulatory mechanism was investigated using luciferase reporter assays. Genotyping and Western blotting demonstrated loss of wild-type Per2 transcript and markedly reduced PER2 protein in Per2 Del4-6 mice. Hepatic expression of a plenty of drug-metabolizing genes (including Cyp2a4/2a5, Cyp2b10, Ugt1a1, Ugt1a9, Ugt2b36, Sult1a1 and Sult1e1) were altered (and majority were down-regulated) in Per2 Del4-6 mice. Of note, Cyp2b10, Ugt1a9 and Sult1a1 were three genes considerably affected with reduced expression. Decreased expression of CYP2B10 was translated to reduced metabolism and altered pharmacokinetics of CPA as well as attenuated CPA hepatotoxicity in Per2 Del4-6 mice. Positive regulation of CYP2B10 by PER2 was further confirmed in both Hepa-1c1c7 and AML-12 cells. Based on luciferase reporter assays, it was shown that PER2 regulated Cyp2b10 transcription in a REV-ERBα-dependent manner. REV-ERBα was negatively regulated by PER2 (increased REV-ERBα expression in Per2 Del4-6 mice) and itself was also a repressor of CYP2B10. In conclusion, PER2 positively regulates CYP2B10 expression and activity in mouse liver through inhibiting its repressor REV-ERBα.

Time-Restricted Feeding in Mice Prevents the Disruption of the Peripheral Circadian Clocks and Its Metabolic Impact during Chronic Jetlag.

We used time-restricted feeding (TRF) to investigate whether microbial metabolites and the hunger hormone ghrelin can become the dominant entraining factor during chronic jetlag to prevent disruption of the master and peripheral clocks, in order to promote health. Therefore, hypothalamic clock gene and Agrp/Npy mRNA expression were measured in mice that were either chronically jetlagged and fed ad libitum, jetlagged and fed a TRF diet, or not jetlagged and fed a TRF diet. Fecal short-chain fatty acid (SCFA) concentrations, plasma ghrelin and corticosterone levels, and colonic clock gene mRNA expression were measured. Preventing the disruption of the food intake pattern during chronic jetlag using TRF restored the rhythmicity in hypothalamic clock gene mRNA expression of Reverbα but not of Arntl. TRF countered the changes in plasma ghrelin levels and in hypothalamic Npy mRNA expression induced by chronic jetlag, thereby reestablishing the food intake pattern. Increase in body mass induced by chronic jetlag was prevented. Alterations in diurnal fluctuations in fecal SCFAs during chronic jetlag were prevented thereby re-entraining the rhythmic expression of peripheral clock genes. In conclusion, TRF during chronodisruption re-entrains the rhythms in clock gene expression and signals from the gut that regulate food intake to normalize body homeostasis.

Dietary Conjugated Linoleic Acid Modulates the Hepatic Circadian Clock Program via PPARα/REV-ERBα-Mediated Chromatin Modification in Mice.

Scope: Disruptions of circadian rhythm cause metabolic disorders and are closely related to dietary factors. In this study, we investigated the interplays between the dietary conjugated linoleic acid (CLA)-induced hepatic steatosis and the circadian clock regulation, in association with lipid homeostasis.Methods and Results: Exposure of mice to 1.5% dietary CLA for 28 days caused insulin resistance, enlarged livers, caused hepatic steatosis, and increased triglyceride levels. Transcriptional profiling showed that hepatic circadian clock genes were significantly downregulated with increased expression of the negative transcription factor, REV-ERBα. We uncovered that the nuclear receptor (NR) PPARα, as a major target of dietary CLA, drives REV-ERBα expression via its binding to key genes of the circadian clock, including Cry1 and Clock, and the recruitment of histone marks and cofactors. The PPARα or REV-ERBα inhibition blocked the physical connection of this NR pair, reduced the cobinding of PPARα and REV-ERBα to the genomic DNA response element, and abolished histone modifications in the CLA-hepatocytes. In addition, we demonstrated that CLA promotes PPARα driving REV-ERBα transcriptional activity by directly binding to the PPAR response element (PPRE) at the Nr1d1 gene.Conclusions: Our results add a layer to the understanding of the peripheral clock feedback loop, which involves the PPARα-REV-ERBα, and provide guidance for nutrients optimization in circadian physiology.

Abstract MP31: Bmal1 Regulates Production Of Larger Lipoproteins By Modulating Crebh And Apolipoprotein A 4

High plasma lipid and lipoprotein levels are risk factors for various metabolic diseases, such as diabetes, obesity, metabolic syndrome, and atherosclerosis. We previously showed that circadian rhythms regulate plasma lipids, and deregulation of these rhythms cause hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific Bmal1-deficient mice maintained on a chow or a Western diet developed hyperlipidemia, which was denoted by the presence of higher amounts of triglyceride- and apoAIV-rich larger chylomicron and very-low-density lipoprotein, due to overproduction. Bmal1 deficiency decreased Shp and increased MTP, a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates Crebh to modulate apoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh- and apoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on apoAIV expression and lipoprotein size. These studies indicate that regulation of apoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and Rev-erbα interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and apoAIV, via two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia, and synchronization of circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.

Vasopressinergic Activity of the Suprachiasmatic Nucleus and mRNA Expression of Clock Genes in the Hypothalamus-Pituitary-Gonadal Axis in Female Aging.

The important involvement of the suprachiasmatic nucleus (SCN) and the activity of vasopressinergic neurons in maintaining the rhythmicity of the female reproductive system depends on the mRNA transcription-translation feedback loops. Therefore, circadian clock function, like most physiological processes, is involved in the events that determine reproductive aging. This study describes the change of mRNA expression of clock genes, Per2, Bmal1, and Rev-erbα, in the hypothalamus-pituitary-gonadal axis (HPG) of female rats with regular cycle (RC) and irregular cycle (IC), and the vasopressinergic neurons activity in the SCN and kisspeptin neurons in the arcuate nucleus (ARC) of these animals. Results for gonadotropins and the cFos/AVP-ir neurons in the SCN of IC were higher, but kisspeptin-ir was minor. Change in the temporal synchrony of the clock system in the HPG axis, during the period prior to the cessation of ovulatory cycles, was identified. The analysis of mRNA for Per2, Bmal1, and Rev-erbα in the reproductive axis of adult female rodents shows that the regularity of the estrous cycle is guaranteed by alternation in the amount of expression of Bmal1 and Per2, and Rev-erbα and Bmal1 between light and dark phases, which ceases to occur and contributes to determining reproductive senescence. These results showed that the desynchronization between the central and peripheral circadian clocks contributes to the irregularity of reproductive events. We suggest that the feedback loops of clock genes on the HPG axis modulate the spontaneous transition from regular to irregular cycle and to acyclicity in female rodents.

Circadian Clock Genes REV-ERBs Inhibits Granulosa Cells Apoptosis by Regulating Mitochondrial Biogenesis and Autophagy in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is an endocrinopathy with complex pathophysiology that is a common cause of anovulatory infertility in women. Although the disruption of circadian rhythms is indicated in PCOS, the role of the clock in the etiology of these pathologies has yet to be appreciated. The nuclear receptors REV-ERBα and REV-ERBβ are core modulators of the circadian clock and participate in the regulation of a diverse set of biological functions. However, in PCOS, the expression of REV-ERBs and their effects remain unclear. Here, we demonstrate that the levels of REV-ERBα and REV-ERBβ expression were lower in the granulosa cells of PCOS patients than in control subjects. In vitro, we found that the overexpression of REV-ERBα and REV-ERBβ, and their agonist SR9009, promoted the expression of mitochondrial biosynthesis genes PGC-1α, NRF1, and TFAM and inhibited autophagy in KGN cells. Our results also indicate that REV-ERBα and REV-ERBβ can inhibit apoptosis in granulosa cells and promote proliferation. Importantly, the REV-ERB agonist SR9009 ameliorates abnormal follicular development by promoting mitochondrial biosynthesis and inhibiting autophagy in a mouse PCOS model. This allows us to speculate that SR9009 has potential as a therapeutic agent for the treatment of PCOS.

Chronoeffects of the Herbal Medicines Puerariae radix and Coptidis rhizoma in Mice: A Potential Role of REV-ERBα.

Identifying drugs with dosing time-dependent effects (chronoeffects) and understanding the underlying mechanisms would help to improve drug treatment outcome. Here, we aimed to determine chronoeffects of the herbal medicines Puerariae radix (PR) and Coptidis rhizoma (CR), and investigate a potential role of REV-ERBα as a drug target in generating chronoeffects. The pharmacological effect of PR on hyperhomocysteinemia in mice was evaluated by measuring total homocysteine, triglyceride levels and lipid accumulation. PR dosed at ZT10 generated a stronger effect on hyperhomocysteinemia than drug dosed at ZT2. Furthermore, PR increased the expression levels of REV-ERBα target genes Bhmt, Cbs and Cth (encoding three key enzymes responsible for homocysteine catabolism), thereby alleviating hyperhomocysteinemia in mice. Moreover, CR attenuated chronic colitis in mice in a dosing time-dependent manner based on measurements of disease activity index, colon length, malondialdehyde/myeloperoxidase activities and IL-1β/IL-6 levels. ZT10 dosing generated a stronger anti-colitis effect as compared to ZT2 dosing. This was accompanied by lower production of colonic inflammatory cytokines (i.e., Nlrp3, IL-1β, IL-6, Tnf-α and Ccl2, REV-ERBα target genes) in colitis mice dosed at ZT10. The diurnal patterns of PR and CR effects were respectively consistent with those of puerarin (a main active constituent of PR, a REV-ERBα antagonist) and berberine (a main active constituent of CR, a REV-ERBα agonist). In addition, loss of Rev-erbα in mice abolished the dosing time-dependency in PR and CR effects. In conclusion, the therapeutic effects of PR and CR depend on dosing time in mice, which are probably attributed to diurnal expression of REV-ERBα as the drug target. Our findings have implications for improving therapeutic outcomes of herbal medicines with a chronotherapeutic approach.

Roles of HDAC3-orchestrated circadian clock gene oscillations in diabetic rats following myocardial ischaemia/reperfusion injury.

The circadian clock is closely related to the development of diabetes mellitus and cardiovascular disease, and disruption of the circadian clock exacerbates myocardial ischaemia/reperfusion injury (MI/RI). HDAC3 is a key component of the circadian negative feedback loop that controls the expression pattern of the circadian nuclear receptor Rev-erbα to maintain the stability of circadian genes such as BMAL1. However, the mechanism by which the HDAC3-orchestrated Rev-erbα/BMAL1 pathway increases MI/RI in diabetes and its relationship with mitophagy have yet to be elucidated. Here, we observed that the clock genes Rev-erbα, BMAL1, and C/EBPβ oscillations were altered in the hearts of rats with streptozotocin (STZ)-induced diabetes, with upregulated HDAC3 expression. Oscillations of Rev-erbα and BMAL1 were rapidly attenuated in diabetic MI/R hearts versus non-diabetic I/RI hearts, in accordance with impaired and rhythm-disordered circadian-dependent mitophagy that increased injury. Genetic knockdown of HDAC3 significantly attenuated diabetic MI/RI by mediating the Rev-erbα/BMAL1 circadian pathway to recover mitophagy. Primary cardiomyocytes with or without HDAC3 siRNA and Rev-erbα siRNA were exposed to hypoxia/reoxygenation (H/R) in vitro. The expression of HDAC3 and Rev-erbα in cardiomyocytes was increased under high-glucose conditions compared with low-glucose conditions, with decreased BMAL1 expression and mitophagy levels. After H/R stimulation, high glucose aggravated H/R injury, with upregulated HDAC3 and Rev-erbα expression and decreased BMAL1 and mitophagy levels. HDAC3 and Rev-erbα siRNA can alleviate high glucose-induced and H/R-induced injury by upregulating BMAL1 to increase mitophagy. Collectively, these findings suggest that disruption of HDAC3-mediated circadian gene expression oscillations induces mitophagy dysfunction, aggravating diabetic MI/RI. Cardiac-specific HDAC3 knockdown could alleviate diabetic MI/RI by regulating the Rev-erbα/BMAL1 pathway to restore the activation of mitophagy.

RORα and REV-ERBα are Associated With Clinicopathological Parameters and are Independent Biomarkers of Prognosis in Gastric Cancer.

Retinoid-related orphan receptor alpha (RORα) and nuclear receptor subfamily 1 group D member 1 (REV-ERBα) play critical roles in many human cancers. Whether RORα and REV-ERBα expression levels are associated with clinical characteristics are poorly understood, and they may be independent predictors of overall survival (OS) and progression-free survival (PFS) in gastric cancer (GC). This study aimed to investigate the correlation of RORα and REV-ERBα expression levels with clinicopathological parameters, OS, and PFS in GC. Immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were employed to assess the expression levels of RORα and REV-ERBα, which were downregulated in GC tissues compared with normal gastric tissues (P < .001; P < .001) and were associated with several clinicopathological parameters, including histological grade (P = .032; P < .001), preoperative carcinoembryonic antigen (CEA) levels (P = .004; P < .001), and tumor-node-metastasis (TNM) stage (P = .015; P < .001). Additionally, low RORα and REV-ERBα expression levels were associated with poor OS and PFS in GC patients, respectively (P < .001; P = .001). Furthermore, univariate Cox regression model analysis showed that histological grade (P < .001; P < .001), preoperative CEA levels (P < .001; P = .001), TNM stage (P < .001; P < .001), lymph node metastasis (P = .002; P = .002), RORα expression levels (P = .001; P < .001), and REV-ERBα expression levels (P < .001; P = .001) were associated with OS and PFS in GC. Multivariate Cox regression model analysis indicated that RORα expression levels and REV-ERBα expression levels are independent factors of OS and PFS in GC. Besides, RORα and REV-ERBα expression may be positively correlated (χ2 = 6.835; P = .009), and GC patients with both high RORα and REV-ERBα expression levels had the best prognosis. In conclusion, RORα and REV-ERBα may coparticipate in tumor activities and show potential to estimate the prognosis of GC.

Polymethoxyflavones in Citrus Regulate Lipopolysaccharide-Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression.

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1β, ASC, and Caspase1, while other flavonoids only affected IL-1β and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

REV-ERBα mediates complement expression and diurnal regulation of microglial synaptic phagocytosis.

The circadian clock regulates various aspects of brain health including microglial and astrocyte activation. Here, we report that deletion of the master clock protein BMAL1 in mice robustly increases expression of complement genes, including C4b and C3, in the hippocampus. BMAL1 regulates expression of the transcriptional repressor REV-ERBα, and deletion of REV-ERBα causes increased expression of C4b transcript in neurons and astrocytes as well as C3 protein primarily in astrocytes. REV-ERBα deletion increased microglial phagocytosis of synapses and synapse loss in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis which was antiphase to REV-ERBα expression. This daily variation in microglial synaptic phagocytosis was abrogated by global REV-ERBα deletion, which caused persistently elevated synaptic phagocytosis. This work uncovers the BMAL1-REV-ERBα axis as a regulator of complement expression and synaptic phagocytosis in the brain, linking circadian proteins to synaptic regulation.

Abstract 17233: Clock Modulating Small Molecule SR9011 Enhances Cell Cycle Arrest in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a rapidly progressing disease of the lung vasculature, characterized by the remodeling of small pulmonary arteries in which pulmonary arterial smooth muscle cells (PASMCs) exhibit a cancer-like phenotype, with uncontrolled replication, self-sustaining growth signals, evasion of growth suppressors and resistance to apoptosis. In healthy cells, cell division is controlled by the circadian clock resulting in timed mitosis and rhythmic DNA replication. This suggests that impaired circadian clock might be involved in the cell cycle disorders seen in PAH. Especially, impaired function of the nuclear receptor Rev-Erbα (a core clock protein) has been associated with circadian clock impairment and cell cycle disorders in cancer. Hypothesis: Rev-Erbα impairment is involved in PAH pathogenesis. Methods &amp; Results: We synchronized (Zeitgeber method using 50% horse serum for 2 hours) human PASMCs isolated from 4 PAH patients and 4 controls, and demonstrated a reduced (p=0.0192) (immunoblot) expression of Rev-Erbα 12 hours post synchronization in PAH-PASMCs compared to controls, associated with increased proliferation (Ki67 assay) and resistance to apoptosis (annexin V assay) (p&lt;0.05), which were reversed by the Rev-Erbα agonist SR9011 (10μM for 24h). RNAseq (n=4/group) demonstrated that most of the downregulated (2-fold cut-off) genes following SR9011 treatments were involved in cytokinesis, meiosis, mitotic processes, cell cycle progression, chromosome segregation and recombination (p&lt;0.05). In vivo , Rev-Erbα was also significantly decreased in male and female monocrotaline rats. SR9011 treatments (n=15/group) for 2 weeks (50mg/kg, i.p.) improved PA hemodynamics ( RVSP:47.80mmHg vs 75.29mmHg \mPAP:26.69mmHg vs 44.36mmHg, p&lt;0.0001); vascular remodeling, decreased proliferation(p=0.0001), increased apoptosis(p=0.0002)) and decreased RV hypertrophy (Fulton Index:0.3869 vs 0.5093 p=0.0006) compare to vehicle treated rats. These results were also repeated in the Fawn Hooded rats model. Conclusion: Our results suggest the involvement of Clock pathways in PAH etiology and thus open new avenue of investigation in PAH and new therapeutic options using clock-modulating small molecules.

Corneal dysfunction precedes the onset of hyperglycemia in a mouse model of diet-induced obesity.

The purpose of this study was to use a mouse model of diet-induced obesity to determine if corneal dysfunction begins prior to the onset of sustained hyperglycemia and if the dysfunction is ameliorated by diet reversal. Six-week-old male C57BL/6 mice were fed a high fat diet (HFD) or a normal diet (ND) for 5-15 weeks. Diet reversal (DiR) mice were fed a HFD for 5 weeks, followed by a ND for 5 or 10 weeks. Corneal sensitivity was determined using aesthesiometry. Corneal cytokine expression was analyzed using a 32-plex Luminex assay. Excised corneas were prepared for immunofluorescence microscopy to evaluate diet-induced changes and wound healing. For wounding studies, mice were fed a HFD or a ND for 10 days prior to receiving a central 2mm corneal abrasion. After 10 days of HFD consumption, corneal sensitivity declined. By 10 weeks, expression of corneal inflammatory mediators increased and nerve density declined. While diet reversal restored nerve density and sensitivity, the corneas remained in a heightened inflammatory state. After 10 days on the HFD, corneal circadian rhythms (limbal neutrophil accumulation, epithelial cell division and Rev-erbα expression) were blunted. Similarly, leukocyte recruitment after wounding was dysregulated and accompanied by delays in wound closure and nerve recovery. In the mouse, obesogenic diet consumption results in corneal dysfunction that precedes the onset of sustained hyperglycemia. Diet reversal only partially ameliorated this dysfunction, suggesting a HFD diet may have a lasting negative impact on corneal health that is resistant to dietary therapeutic intervention.

Rev-erbα regulates hepatic ischemia-reperfusion injury in mice

Hepatic ischemia-reperfusion (I/R) injury is a complex pathophysiological process that often times occurs in liver transplantation, hepatectomy, and ischemic shock. Aberrant activation of inflammatory responses has been implicated in hepatic I/R injury. In this study, we aimed to investigate the role of circadian clock gene Rev-erbα (a well-known regulator of inflammation) in hepatic I/R injury. We first showed that Rev-erbα ablation sensitized mice to hepatic I/R injury as evidenced by higher levels of plasma alanine aminotransferase and aspartate aminotransferase, an increased histological score, as well as enhanced hepatic myeloperoxidase activity in Rev-erbα−/− mice. More severe hepatic I/R injury in Rev-erbα−/− mice was accompanied by higher expression of pro-inflammatory cytokines, exacerbated activation of Nlrp3 inflammasome, and more extensive infiltration of inflammatory cells. Moreover, pharmacological activation of Rev-erbα by SR9009 significantly alleviated the hepatic damage and inflammatory responses. In addition, I/R operation started at ZT18 (corresponding to low Rev-erbα expression) caused more severe liver damage and inflammatory responses in wild-type mice as compared to operation started at ZT6 (corresponding to high Rev-erbα expression), supporting a protective effect of Rev-erbα on hepatic I/R injury. Collectively, Rev-erbα protects hepatic I/R injury probably via repression of inflammatory responses, and targeting Rev-erbα may be a promising approach for management of hepatic I/R injury.

Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals

ObjectiveSkeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. Twenty-four-hour rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans remains unknown. MethodsTwelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at 5 time points (8 AM, 1 PM, 6 PM, 11 PM, 4 AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry. ResultsMitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBα showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level, resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease during the night, indicating metabolic inflexibility. ConclusionsMitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed. ClinicalTrials.gov IDNCT03733743.