BMAL1 Protein Levels Research Articles

Time of day-dependent alterations of ferroptosis in LPS-induced myocardial injury via Bmal-1/AKT/ Nrf2 in rat and H9c2 cell

BackgroundOne of the most prevalent causes of death in sepsis is sepsis-induced cardiomyopathy (SICM). Circadian disruption is involved in the progress of sepsis. However, the molecular mechanism remains unclear. MethodsHere, we built LPS-induced SICM in-vivo and in-vitro models. LPS was administrated at the particular Zeitgeber times (ZT), ZT4-ZT10-ZT16-ZT22 and ZT10-ZT22 in vivo and vitro experiments, respectively. ResultsIn vivo experiment, injection of LPS at ZT10 induced higher infiltration of inflammatory cells and content of intracellular Fe2+, and lower level of Glutathione peroxidase 4 (GPX4) and cardiac function than other ZTs (P < 0.05), which indicated that myocardial ferroptosis in septic rat presented a time of day-dependent manner. Bmal-1 protein and mRNA levels of injection of LPS at ZT10 were lower than those at other three ZTs (P < 0.05). The ratios of pAKT/AKT at ZT4 and ZT10 LPS injection were lower than those at ZT16 and ZT22 (P < 0.05). Nrf2 protein levels at ZT10 LPS injection were lower than those at other three ZTs (P < 0.05). These results indicated that the circadian of Bmal-1 and its downstream AKT/Nrf2 pathway in rat heart were inhibited under SICM condition. Consistent with in-vivo experiment, we found LPS could significantly reduce the expressions of Bmal-1 protein and mRNA in H9c2 cell. Up-regulation of Bmal-1 could reduce the cell death, oxidative stress, ferroptosis and activation of AKT/Nrf2 pathway at both ZT10 and ZT22 LPS administration. Conversely, its down-regulation presented opposite effects. AKT siRNA could weaken the effect of Bmal-1 pcDNA. ConclusionFerroptosis presented the time of day-dependent manners via Bmal-1/AKT/Nrf2 in vivo and vitro models of SICM.

Melatonin attenuates chronic sleep deprivation-induced cognitive deficits and HDAC3-Bmal1/clock interruption.

Sleep is predicted as a key modulator of cognition, but the underlying mechanisms are poorly understood. In this study, we investigated the effects of melatonin on chronic rapid eye movement sleep deprivation (CRSD)-induced cognitive impairment and circadian dysfunction in rat models. Thirty-six Sprague-Dawley male rats were divided into three groups: CRSD with saline treatment, CRSD with chronic melatonin injection (20 mg/kg/day), and non-sleep-deprived control. The cognitive behavioral tests as well as the expression of clocks and HDAC3 were evaluated in all groups. CRSD significantly reduced recognition index in novel object location, increased escape latency and distance traveling in Morris water maze while melatonin treatment attenuated CRSD-induced hippocampal-dependent spatial learning and memory deficits. Furthermore, the mRNAs of brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1(Bmal1) and circadian locomotor output cycles kaput (Clock) were globally down-regulated by CRSD with constant intrinsic oscillation in both hippocampus and peripheral blood. The protein levels of hippocampal Bmal1, Clock, and HDAC3 were also remarkably down-regulated following CRSD. Melatonin treatment reversed CRSD-induced alterations of Bmal1/Clock and HDAC3 on both mRNA levels and protein levels. Our data indicate that melatonin treatment attenuates CRSD-induced cognitive impairment via regulating HDAC3-Bmal1/Clock interaction. These findings explore a broader understanding of the relationship between sleep and cognition and provide a potential new therapeutic target for cognitive impairment.

Dihydroisotanshinone I and BMAL-SIRT1 Pathway in an In Vitro 6-OHDA-Induced Model of Parkinson’s Disease

Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson’s disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of BMAL1 and SIRT1 in the 6-OHDA-treated SH-SY5Y cells. Additionally, the SIRT1 inhibitor attenuated the effect of DT on BMAL1 and reduced the cell viability. The DT and SIRT1 activators activated SIRT1 and BMAL1, and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed.

0128 Muscle injury induces an increase in total and NREM sleep time

Abstract Introduction This study describes macro- and micro- sleep responses to a myotoxic skeletal muscle injury and investigates possible mechanisms. Methods We recorded the electroencephalogram(EEG)/electromyogram(EMG) of 24 wistar rats before and after induction of Tibialis Anterior muscle injury (n=8 per group: control, control +buprenorphine and injured). A top-down analysis of sleep characteristics was processed from total sleep time (TST), sleep stages, sleep stability, spectral-analysis, and spindles. To further investigate the mechanisms involved, we analyzed the protein level of sleep regulatory molecules including TNF-α, IL-1β, IGF-1, BMAL1 in plasma, frontal cortex, hippocampus, and tibialis anterior, collected at Day +2 after injury from non-EEG/EMG implanted rats. Results Muscle injury induces a significant increase in total sleep time at 48 and 72h post-injury, specific to NREM sleep. These increases occur during the dark period and are associated with higher stability of sleep over 24h, without change in the different power/frequency spectral bands of NREM/REM sleep. There was no corresponding sleep increase in slow-wave activity or spindle density, nor were there changes in brain levels of the sleep-regulating proinflammatory cytokine IL-1β, which is otherwise involved in the local response to injury. Conversely, decreased protein levels of brain IGF-1 and muscle BMAL1, a core circadian clock gene, after injury may play a role in increased sleep time. Conclusion Muscle injury induces an increase in total sleep time at 48- and 72-hours post-injury, specific to NREM sleep during the dark period in rats and is associated with higher sleep stability over 24 hours. Support (if any)

Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare.

Gout is caused by monosodium urate (MSU) crystal deposition within joints. This leads to acute episodes of inflammation ("gout flares") driven by NLRP3 inflammasome activation in macrophages. Gout flares are frequently present during late night/early morning. The reason for this timing is unclear. Recent evidence suggests the NLRP3 inflammasome is under circadian control. The purpose of this study was to determine whether MSU crystals cause changes in the circadian clock in macrophages leading to time-of-day differences in NLRP3 inflammasome activation. Levels of circadian clock components were measured in undifferentiated "monocytic" and PMA-differentiated "macrophagic" THP-1 cells cultured with/without MSU crystals. Caspase-1 activity was measured to assess NLRP3 inflammasome activity. MSU crystal exposure resulted in minimal effects on clock genes in THP-1 monocytes but BMAL1, CRY1, PER2, and REV-ERBα showed altered expression with reduced protein levels of BMAL1 and REV-ERBα in THP-1 macrophages. REV-ERBα activation or BMAL1 over-expression resulted in reduced MSU crystal-induced caspase-1 activity. BMAL1 knockdown resulted in a further increase in MSU crystal-induced caspase-1 activity, but only at times of day when BMAL1 levels were naturally high. MSU crystal-induced NLRP3 inflammasome activation was greatest at the time of day when BMAL1 levels were naturally low. MSU crystals alter the expression of circadian clock components in THP-1 macrophages leading to loss of BMAL1 and REV-ERBα-mediated repression of NLRP3 inflammasome activity and time-of-day differences in susceptibility to inflammasome activation. Our findings suggest that the nocturnal risk of gout flare is at least partially a consequence of altered circadian control of immune cell function.

Berberine Ameliorates Metabolic-Associated Fatty Liver Disease Mediated Metabolism Disorder and Redox Homeostasis by Upregulating Clock Genes: Clock and Bmal1 Expressions.

Metabolic-associated fatty liver disease (MAFLD) is one of the most common chronic liver diseases, which in turn triggers mild inflammation, metabolic dysfunction, fibrosis, and even cancer. Accumulating evidence has suggested that Berberine (BBR) could significantly improve MAFLD progression. Clock and Bmal1 as heterodimer proteins highly participated in the development of MAFLD, but whether BBR targets Clock and Bmal1 in MAFLD remains poorly understood. The result suggested that the protein levels of Clock and Bmal1 were decreased in MAFLD mice, which was negatively correlated with elevated reactive oxygen species (ROS) accumulation, the H2O2 level, liver inflammation, metabolic dysfunction, and insulin resistance. The mRNA and protein levels of Clock and Bmal1 were also decreased in glucosamine-induced HepG2 cells, which were are negatively related to glucose uptake, the ROS level, and the H2O2 level. More importantly, Bmal1 siRNA could mimic the effect of glucosamine in HepG2 cells. Interestingly, Berberine (BBR) could rescue metabolism disorder and redox homeostasis through enhancing Clock and Bmal1 expression in vivo and in vitro. Therefore, BBR might be an effective natural compound for alleviating redox homeostasis, metabolism disorder, and liver pathological changes in MAFLD by activating Clock and Bmal1 expression.

Fluoxetine Decreases Phagocytic Function via REV-ERBα in Microglia.

Although fluoxetine (FLX) is a commonly used drug in psychiatric disorders, such as major depressive disorder, anxiety disorder, panic disorder, and obsessive-compulsive disorder, the mechanism by which FLX exerts its therapeutic effect is not completely understood. In this study, we aimed to determine the possible mechanism by which FLX focuses on microglial phagocytosis. FLX reduced phagocytic function in BV2 cells and increased REV-ERBα without affecting other microglia-related genes, such as inflammation and phagocytosis. Although FLX did not change BMAL1 protein levels, it restricted the nucleocytoplasmic transport (NCT) of BMAL1, leading to its cytosolic accumulation. REV-ERBα antagonist SR8278 rescued the decreased phagocytic activity and restricted NCT of BMAL1. We also found that REV-ERBα mediates the effect of FLX via the inhibition of phospho-ERK (pERK). The ERK inhibitor FR180204 was sufficient to reduce phagocytic function in BV2 cells and restrict the NCT of BMAL1. These results were recapitulated in the primary microglia. In conclusion, we propose that FLX decreases phagocytic function and restricts BMAL1 NCT via REV-ERBα. In addition, ERK inhibition mimics the effects of FLX on microglia.

Haploinsufficiency of a Circadian Clock Gene Bmal1 (Arntl or Mop3) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice.

Approximately 50–80% of children with autism spectrum disorders (ASDs) exhibit sleep problems, but the contribution of circadian clock dysfunction to the development of ASDs remains largely unknown. The essential clock gene Bmal1 (Arntl or Mop3) has been associated with human sociability, and its missense mutation is found in ASD. Our recent study found that Bmal1-null mice exhibit a variety of autism-like phenotypes. Here, we further investigated whether an incomplete loss of Bmal1 function could cause significant autism-like behavioral changes in mice. Our results demonstrated that heterozygous Bmal1 deletion (Bmal1+/−) reduced the Bmal1 protein levels by ~50–75%. Reduced Bmal1 expression led to decreased levels of clock proteins, including Per1, Per2, Cry 1, and Clock but increased mTOR activities in the brain. Accordingly, Bmal1+/− mice exhibited aberrant ultrasonic vocalizations during maternal separation, deficits in sociability and social novelty, excessive repetitive behaviors, impairments in motor coordination, as well as increased anxiety-like behavior. The novel object recognition memory remained intact. Together, these results demonstrate that haploinsufficiency of Bmal1 can cause autism-like behavioral changes in mice, akin to those identified in Bmal1-null mice. This study provides further experimental evidence supporting a potential role for disrupted clock gene expression in the development of ASD.

Circadian molecular clock disruption in chronic pulmonary diseases.

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future.

RCAN1 knockout and overexpression recapitulate an ensemble of rest-activity and circadian disruptions characteristic of Down syndrome, Alzheimer’s disease, and normative aging

BackgroundRegulator of calcineurin 1 (RCAN1) is overexpressed in Down syndrome (DS), but RCAN1 levels are also increased in Alzheimer’s disease (AD) and normal aging. AD is highly comorbid among individuals with DS and is characterized in part by progressive neurodegeneration that resembles accelerated aging. Importantly, abnormal RCAN1 levels have been demonstrated to promote memory deficits and pathophysiology that appear symptomatic of DS, AD, and aging. Anomalous diurnal rest-activity patterns and circadian rhythm disruptions are also common in DS, AD, and aging and have been implicated in facilitating age-related cognitive decline and AD progression. However, no prior studies have assessed whether RCAN1 dysregulation may also promote the age-associated alteration of rest-activity profiles and circadian rhythms, which could in turn contribute to neurodegeneration in DS, AD, and aging.MethodsThe present study examined the impacts of RCAN1 deficiency and overexpression on the photic entrainment, circadian periodicity, intensity and distribution, diurnal patterning, and circadian rhythmicity of wheel running in young (3–6 months old) and aged (9–14 months old) mice of both sexes.ResultsWe found that daily RCAN1 levels in the hippocampus and suprachiasmatic nucleus (SCN) of light-entrained young mice are generally constant and that balanced RCAN1 expression is necessary for normal circadian locomotor activity rhythms. While the light-entrained diurnal period was unaltered, RCAN1-null and RCAN1-overexpressing mice displayed lengthened endogenous (free-running) circadian periods like mouse models of AD and aging. In light-entrained young mice, RCAN1 deficiency and overexpression also recapitulated the general hypoactivity, diurnal rest-wake pattern fragmentation, and attenuated amplitudes of circadian activity rhythms reported in DS, preclinical and clinical AD, healthily aging individuals, and rodent models thereof. Under constant darkness, RCAN1-null and RCAN1-overexpressing mice displayed altered locomotor behavior indicating circadian clock dysfunction. Using the Dp(16)1Yey/+ (Dp16) mouse model for DS, which expresses three copies of Rcan1, we found reduced wheel running activity and rhythmicity in both light-entrained and free-running young Dp16 mice like young RCAN1-overexpressing mice. Critically, these diurnal and circadian deficits were rescued in part or entirely by restoring Rcan1 to two copies in Dp16 mice. We also found that RCAN1 deficiency but not RCAN1 overexpression altered protein levels of the clock gene Bmal1 in the SCN.ConclusionsCollectively, this study’s findings suggest that both loss and aberrant gain of RCAN1 precipitate anomalous light-entrained diurnal and circadian activity patterns emblematic of DS, AD, and possibly aging.

Day-night oscillations of cognitive functions, TNF alpha and clock -related factors expression are modified by an intracerebroventricular injection of amyloid beta peptide in rat

Alzheimer's disease (AD) is the most common form of dementia characterized by a gradual impairment in cognitive functions. Recent research have shown that TNF-α is a proinflammatory cytokine implicated in the pathogenesis of neurodegenerative diseases, such as AD. Besides cognitive deficit, AD patients show alterations in their circadian rhythms. The objective of this work was to investigate the effects of an intracerebroventricular injection of Aß aggregates on temporal patterns of cognitive functions and on daily rhythms of Aβ, TNFα, BMAL1 and RORα protein levels in the rat prefrontal cortex. Four-month-old males Holtzman rats were used in this study. Groups were defined as: control and Aβ-injected rats. Rats were maintained under 12h-light:12h-dark throughout the entire experimental period. Prefrontal cortex samples were isolated every 4 h during a 24h period. Our results demonstrated that an intracerebroventricular injection of Aß aggregates impaired learning and memory in rats at ZT 2 and ZT 14 and modified daily patterns of Aβ, TNFα, and clock-related factors in the rat prefrontal cortex. Our findings showed that the increase of Aß altered temporal patterns of TNFα, and, consequently, induced alterations in daily rhythms of clock-related factors, affecting the cognitive performance of animals with Alzheimer's.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm–Controlled Gene Expression in Mice

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Aging disrupts the temporal organization of antioxidant defenses in theheart of male ratsand phase shifts circadian rhythms of systolic blood pressure.

Aging is one of the main risk factors for cardiovascular diseases, and oxidative stress is a key element responsible for the development of age-related pathologies. In addition, the alteration of circadian rhythms also contributes to cardiovascular pathology, but the underlying mechanisms are not well defined. We investigated the aging consequences on the temporal patterns of antioxidant defenses, the molecular clock machinery, and the blood pressure, in the heart of male rats maintained under constant darkness (free running) conditions. Male Holtzman rats from young adult (3-month-old) and older (22-month-old) groups were maintained under constant darkness (12-h dark:12-h dark, DD) condition during fifteen days before the experiment. After the DD period, heart ventricle samples were isolated every 4-h throughout a 24-h period. We observed circadian rhythms of catalase (CAT) and glutathione peroxidase (GPx) mRNA expression, as well as ultradian rhythms of Nrf2 mRNA levels, in the heart of young adult rats. We also found circadian oscillations of CAT and GPx enzymatic activities, reduced glutathione (GSH) and BMAL1 protein in the same group. Interestingly, aging abolished the rhythms of CAT and GPx enzymatic activities, phase-shifted the rhythm's acrophases of GSH and BMAL1 protein levels and turned circadian the ultradian oscillation of Nrf2 expression. Moreover, aging phase-shifted the circadian pattern of systolic blood pressure. In conclusion, aging modifies the temporal organization of antioxidant defenses and blood pressure, probably, as a consequence of a disruption in the circadian rhythm of the clock's transcriptional regulator, BMAL1, in heart.

Alterations of sleep quality and circadian rhythm genes expression in elderly thyroid nodule patients and risks associated with thyroid malignancy

To study the alterations of sleep quality and circadian rhythm genes expressions upon elderly thyroid nodule patients, the risk factors associated with thyroid malignancies, and the potential relationship involved. The elderly people enrolled in our study were divided into three groups according to the thyroid histopathology: malignant nodule group, benign nodule group, and normal group, and the clinical data and sleep quality were collected. Among the patients of surgery, 56 fresh thyroid tissues were collected for real-time PCR, immunohistochemistry and western blotting analysis of CLOCK, BMAL1, CRYs and PERs. Poor sleep quality, sleep latency and daytime dysfunction were the independent risk factors of malignant nodule after adjusted by other impacts. The expression levels of CLOCK, BMAL1 and PER2 in thyroid malignant group were significantly higher than benign and normal groups, while CRY2 was decreased, p < 0.05. In addition, CLOCK and BMAL1 protein levels were positively correlated with PSQI of corresponding patients and CRY2 was negatively correlated. Circadian rhythm genes mainly altered in malignant nodules, and sleep disorders may be involved in the occurrence of elderly thyroid malignancy through the high expressions of CLOCK and BMAL1, and low expression of CRY2.

Obese Neotomodon alstoni mice exhibit sexual dimorphism in the daily profile of circulating melatonin and clock proteins PER1 and BMAL1 in the hypothalamus and peripheral oscillators

ABSTRACT Obesity is a global health threat and a risk factor for several metabolic conditions. Though circadian dysfunction has been considered among the multiple causes of obesity, little work has been done to explore the relationship between obesity, circadian dysfunction, and sexual dimorphism. The Neotomodon alstoni mouse is a suitable model for such research. This study employed N. alstoni mice in a chronobiological analysis to determine whether there is circadian desynchronization of relative PER1 and BMAL1 protein levels in the hypothalamus, liver, visceral white adipose tissue, kidney, and heart. It also compared differences between sexes and lean and obese N. alstoni adult mice, by recording behavior and daily circulating serum melatonin as markers of circadian output. We found that obese mice display reduced locomotor activity. Additionally, Cosinor analyses of the relative expression of PER1 and BMAL1 show differences between lean and obese mice in a sex-linked manner. The PER1 24 h rhythm was absent in all tissues of obese males and significant in the tissues of obese females. The BMAL1 24 h rhythm also was significant in most of the tissues tested in lean males, whereas it was significant and shifted the acrophase (peak time of rhythm) in most of the tissues in obese females. Both lean male and female mice showed a rhythmic 24 h pattern of circulating serum melatonin. This daily profile was not only absent in obese mice of both sexes but showed sexual dimorphism. Obese male mice showed lower circulating levels of melatonin compared to lean male mice, but they were higher in obese females compared to lean females. Our results suggest that obesity in N. alstoni is associated with an internal circadian desynchronization in a sex-dependent manner. Overall, this study reinforces the need for further research on the neuroendocrinology of obesity and circadian rhythms using this biological model.

Zearalenone perturbs the circadian clock and inhibits testosterone synthesis in mouse Leydig cells

ABSTRACT Zearalenone (ZEA), a mycotoxin, is known to impair reproductive capability by disrupting the synthesis and secretion of testosterone by Leydig cells (LCs), although the mechanism is unknown. Robust rhythmicity of circadian clock and steroidogenic genes were identified in LCs. The aim of this study was to examine whether ZEA significantly attenuated the transcription of core clock genes (Bmal1, Dbp, Per2, and Nr1d1) as well as steroidogenic genes (StAR, Hsd3b2, and Cyp11a1) in mouse testis Leydig cell line (TM3). Western blotting confirmed declines in BMAL1, NR1D1, and StAR protein levels. ZEA also suppressed secreted testosterone levels. In primary LCs, isolated from PER2::LUCIFERASE reporter gene knock in mice, ZEA diminished the amplitude of PER2::LUC expression, and induced a phase shift and period extension. In primary LCs, ZEA also suppressed the expression levels of core clock and steroidogenic genes, reduced protein levels of BMAL1, and decreased testosterone secretion. In vivo expression of core clock and steroidogenic genes were reduced in testes of mice exposed to ZEA for 1 week leading to decreased serum testosterone levels. In summary, data suggest that ZEA may impair testosterone synthesis through attenuation of the circadian clock in LCs culminating in reproductive dysfunction in male mammals .

Expression of circadian clock genes during differentiation of rat dental papilla cells in vitro

ABSTRACT The circadian clock is involved in many physiological processes including teeth development. The expression of circadian core clock genes was observed in various tissues, but their role in odontoblasts is unclear. The aim of this study was to investigate the circadian core clock gene expressions of dental papilla cells (DPCs) during the odontogenic differentiation. Primary DPCs were obtained from the first molar dental papilla of neonatal rats and cultured in osteogenic (OS) medium for differentiation in vitro. Reverse transcription and quantitative PCR was performed to assess the mRNA levels of the circadian core clock genes Bmal1, Per2, Cry1, Clock, Rev-erb α and Ror α. Western blotting was performed to assess the protein levels of BMAL1 and PER2. The results showed that apparent oscillatory expression patterns of Per2, Cry1, Rev-erb α could be observed in DPCs, whether differentiated or not. The protein level of BMAL1 was significantly increased and PER2 was decreased during odontogenic differentiation. Taken together, these findings demonstrate that circadian rhythmicity of known core clock genes exist in cultured DPCs in vitro, and the expression of genes change during differentiation process.

The circadian clock protects against acute radiation-induced dermatitis

Radiation-induced dermatitis is a common occurrence in cancer patients undergoing radiation therapy (RT) and is caused when ionizing radiation (IR) induces DNA strand breaks in skin cells. The wide use of RT in cancer treatments makes it important to minimize RT-induced toxicities including radiodermatitis. This study sought to determine if the circadian clock plays a protective role in minimizing radiodermatitis. We treated mice in control (Day Shift), environmentally-disrupted (Rotating Shift) and genetically-disrupted (Per 1/2−/−) circadian conditions with 6 Gy of IR to the whole body. There was a significantly increased number of radiodermatitis spots on mice with circadian clock disruption compared to control mice. Additionally, circadian clock disrupted mice exhibited reduced protein levels of Bmal1, a phenomenon that sensitized circadian synchronized keratinocytes to IR-induced DNA damage. Furthermore, the skin phenotype results corresponded with significantly reduced body weights and increased genomic DNA damage in blood cells of mice with clock disruption compared to control mice. These findings suggest that the circadian clock plays a protective role in IR-induced DNA damage and skin toxicity, possibly through BMAL1-dependent mechanisms, and potentially impacts RT-associated radiodermatitis in cancer patients.

The E3 ubiquitin ligase STUB1 attenuates cell senescence by promoting the ubiquitination and degradation of the core circadian regulator BMAL1

Cell senescence is one of the most important processes determining cell fate and is involved in many pathophysiological conditions, including cancer, neurodegenerative diseases, and other aging-associated diseases. It has recently been discovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1 or CHIP) is up-regulated during the senescence of human fibroblasts and modulates cell senescence. However, the molecular mechanism underlying STUB1-controlled senescence is not clear. Here, using affinity purification and MS-based analysis, we discovered that STUB1 binds to brain and muscle ARNT-like 1 (BMAL1, also called aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL)). Through biochemical experiments, we confirmed the STUB1-BMAL1 interaction, identified their interaction domains, and revealed that STUB1 overexpression down-regulates BMAL1 protein levels through STUB1's enzymatic activity and that STUB1 knockdown increases BMAL1 levels. Further experiments disclosed that STUB1 enhances BMAL1 degradation, which is abolished upon proteasome inhibition. Moreover, we found that STUB1 promotes the formation of Lys-48-linked polyubiquitin chains on BMAL1, facilitating its proteasomal degradation. Interestingly, we also discovered that oxidative stress promotes STUB1 nuclear translocation and enhances its co-localization with BMAL1. STUB1 expression attenuates hydrogen peroxide-induced cell senescence, indicated by a reduced signal in senescence-associated β-gal staining and decreased protein levels of two cell senescence markers, p53 and p21. BMAL1 knockdown diminishes this effect, and BMAL1 overexpression abolishes STUB1's effect on cell senescence. In summary, the results of our work reveal that the E3 ubiquitin ligase STUB1 ubiquitinates and degrades its substrate BMAL1 and thereby alleviates hydrogen peroxide-induced cell senescence.

A Non-canonical Function of BMAL1 Metabolically Limits Obesity-Promoted Triple-Negative Breast Cancer.

SummaryThe epidemiological association between disrupted circadian rhythms and metabolic diseases is implicated in increased risk of human breast cancer and poor therapeutic outcomes. To define a metabolic phenotype and the underlying molecular mechanism, we applied chronic insulin treatment (CIT) to an in vitro model of triple-negative breast cancer to directly address how BMAL1, a key circadian transcription factor, regulates cancer cell respiration and governs tumor progression. At the cellular level, BMAL1 suppresses the flexibility of mitochondrial substrate usage and the pyruvate-dependent mitochondrial respiration induced by CIT. We established an animal model of diet-induced obesity/hyperinsulinemia and observed that BMAL1 functions as a tumor suppressor in obese, but not lean, mice. Downregulation of BMAL1 is associated with higher risk of metastasis in human breast tumors. In summary, loss of BMAL1 in tumors confers advantages to cancer cells in both intrinsic mitochondrial metabolism and extrinsic inflammatory tumor microenvironment during pre-diabetic obesity/hyperinsulinemia.