Muscle ARNT-like Research Articles

Sex differences in the adrenal circadian clock: A role for BMAL1 in the regulation of urinary aldosterone excretion and renal electrolyte balance in mice.

Brain and muscle ARNT-Like 1 (BMAL1) is a circadian clock transcription factor that regulates physiological functions. Male adrenal-specific Bmal1 (ASCre/+::Bmal1) KO mice displayed blunted serum corticosterone rhythms, altered blood pressure rhythm, and altered timing of eating, but there is a lack of knowledge in females. This study investigates the role of adrenal BMAL1 in renal electrolyte handling and urinary aldosterone levels in response to low salt in male and female mice. Mice were placed in metabolic cages to measure 12-hour urinary aldosterone after a standard diet and 7 days low salt diet, as well as daily body weight, 12-hour food and water intake, and renal sodium and potassium balance. Adrenal glands and kidneys were collected at ZT0 or ZT12 to measure expression of aldosterone synthesis genes and clock genes. Compared to littermate controls, ASCre/+::Bmal1 KO male and female mice displayed increased urinary aldosterone in response to a low salt diet, although mRNA expression of aldosterone synthesis genes was decreased. Timing of food intake was altered in ASCre/+::Bmal1 KO male and female mice, with a blunted night/day ratio. ASCre/+::Bmal1 KO female mice displayed decreases in renal sodium excretion in response to low salt, but both male and female KO mice had changes in sodium balance that were time-of-day-dependent. In addition, sex differences were found in adrenal and kidney clock gene expression. Notably, this study highlights sex differences in clock gene expression that could contribute to sex differences in physiological functions.

The Influence of Circadian Rhythms on DNA Damage Repair in Skin Photoaging.

Circadian rhythms, the internal timekeeping systems governing physiological processes, significantly influence skin health, particularly in response to ultraviolet radiation (UVR). Disruptions in circadian rhythms can exacerbate UVR-induced skin damage and increase the risk of skin aging and cancer. This review explores how circadian rhythms affect various aspects of skin physiology and pathology, with a special focus on DNA repair. Circadian regulation ensures optimal DNA repair following UVR-induced damage, reducing mutation accumulation, and enhancing genomic stability. The circadian control over cell proliferation and apoptosis further contributes to skin regeneration and response to UVR. Oxidative stress management is another critical area where circadian rhythms exert influence. Key circadian genes like brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) modulate the activity of antioxidant enzymes and signaling pathways to protect cells from oxidative stress. Circadian rhythms also affect inflammatory and immune responses by modulating the inflammatory response and the activity of Langerhans cells and other immune cells in the skin. In summary, circadian rhythms form a complex defense network that manages UVR-induced damage through the precise regulation of DNA damage repair, cell proliferation, apoptosis, inflammatory response, oxidative stress, and hormonal signaling. Understanding these mechanisms provides insights into developing targeted skin protection and improving skin cancer prevention.

Advancing Clinical Response Against Glioblastoma: Evaluating SHP1705 CRY2 Activator Efficacy in Preclinical Models and Safety in Phase I Trials.

It has been reported that circadian clock components, Brain and Muscle ARNT-Like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK), are uniquely essential for glioblastoma (GBM) stem cell (GSC) biology and survival. Consequently, we developed a novel Cryptochrome (CRY) activator SHP1705, which inhibits BMAL1-CLOCK transcriptional activity. We analyzed buffy coats isolated from Phase 1 clinical trial subjects' blood to assess any changes to circadian, housekeeping, and blood transcriptome-based biomarkers following SHP1705 treatment. We utilized GlioVis to determine which circadian genes are differentially expressed in non-tumor versus GBM tissues. We employed in vitro and in vivo methods to test the efficacy of SHP1705 against patient-derived GSCs and xenografts in comparison to earlier CRY activator scaffolds. Additionally, we applied a novel-REV-ERB agonist SR29065, which inhibits BMAL1 transcription, to determine whether targeting both negative limbs of the circadian transcription-translation feedback loop (TTFL) would yield synergistic effects against various GBM cells. SHP1705 is safe and well-tolerated in Phase I clinical trials. SHP1705 has increased selectivity for the CRY2 isoform and potency against GSC viability compared to previously published CRY activators. SHP1705 prolonged survival in mice bearing GBM tumors established with GSCs. When combined with the novel REV-ERB agonist SR29065, SHP1705 displayed synergy against multiple GSC lines and differentiated GSCs (DGCs). These demonstrate the efficacy of SHP1705 against GSCs, which pose for GBM patient outcomes. They highlight the potential of novel circadian clock compounds in targeting GBM as single agents or in combination with each other or current standard-of-care. SHP1705 is a novel CRY2 activator that has shown success in Phase 1 safety trialsSHP1705 has a significantly improved efficacy against GSCs and GBM PDX tumorsNovel REV-ERB agonist SR29065 and SHP1705 display synergistic effects against GSCs. CRY2 is decreased in GBM tissues compared to CRY1 suggesting that promoting CRY2 activity will be an efficacious GBM treatment paradigm. SHP1705, a CRY2 activator that has shown success in Phase 1 safety trials, has significantly improved preclinical efficacy. Novel REV-ERB agonist SR29065 displays synergistic effects against diverse GBM cells.

Circadian organization of clock factors, antioxidant defenses, and cognitive genes expression, is lost in the cerebellum of aged rats. Possible targets of therapeutic strategies for the treatment of age-related cerebellar disorders

Aging is a major risk factor for cognitive deficits, impaired locomotion, and gait disorders. Although oxidative stress and circadian disruption are involved in both normal aging and the pathogenesis of age‐associated diseases, just a very few studies explore the consequences of aging on circadian rhythms in the cerebellum. Here, we investigated age-dependent changes in the circadian organization of the molecular clock, antioxidant defenses and synaptic plasticity-related factors, in the rat cerebellum, and discussed the impact of that altered temporal organization on the cognitive function of this brain area. Particularly, we examined the circadian patterns of Brain and muscle ARNT-like 1 (BMAL1) protein levels, Glutathione peroxidase 4 (GPx4) gene expression, GPx and Catalase (CAT) enzymes activity, reduced glutathione (GSH) levels, and the Brain-derived neurotrophic factor (Bdnf) and its Tyrosine kinase receptor B (TrkB) circadian expression. Endogenously-driven circadian rhythms of BMAL1, GPx4, CAT, GSH, and Bdnf/TrkB factors, were observed in the young rat cerebellum. The rhythms’ acrophases show a circadian organization that might be crucial for the daily cerebellar-dependent cognitive functions. Notably, aging disrupted circadian rhythms and the temporal organization of BMAL1, antioxidant defenses, and cognitive Bdnf/TrkB gene expression. Increased oxidative stress and disruption of clock-controlled rhythms during aging, might precede and cause the loss of circadian organization in the aged cerebellum. We expect our results highlight circadian rhythms of the studied factors as new targets for the treatment of age-dependent cerebellar disorders.

Circadian disruption, clock genes, and metabolic health.

A growing body of research has identified circadian-rhythm disruption as a risk factor for metabolic health. However, the underlying biological basis remains complex, and complete molecular mechanisms are unknown. There is emerging evidence from animal and human research to suggest that the expression of core circadian genes, such as circadian locomotor output cycles kaput gene (CLOCK), brain and muscle ARNT-Like 1 gene (BMAL1), period (PER), and cyptochrome (CRY), and the consequent expression of hundreds of circadian output genes are integral to the regulation of cellular metabolism. These circadian mechanisms represent potential pathophysiological pathways linking circadian disruption to adverse metabolic health outcomes, including obesity, metabolic syndrome, and type 2 diabetes. Here, we aim to summarize select evidence from in vivo animal models and compare these results with epidemiologic research findings to advance understanding of existing foundational evidence and potential mechanistic links between circadian disruption and altered clock gene expression contributions to metabolic health-related pathologies. Findings have important implications for the treatment, prevention, and control of metabolic pathologies underlying leading causes of death and disability, including diabetes, cardiovascular disease, and cancer.

The impact of aerobic exercise timing on BMAL1 protein expression and antioxidant responses in skeletal muscle of mice

It is well known that the adaptations of muscular antioxidant system to aerobic exercise depend on the frequency, intensity, duration, type of the exercise. Nonetheless, the timing of aerobic exercise, related to circadian rhythms or biological clock, may also affect the antioxidant defense system, but its impact remains uncertain. Bain and muscle ARNT-like 1 (BMAL1) is the core orchestrator of molecular clock, which can maintain cellular redox homeostasis by directly controlling the transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2). So, our research objective was to evaluate the impacts of aerobic exercise training at various time points of the day on BMAL1 and NRF2-mediated antioxidant system in skeletal muscle. C57BL/6J mice were assigned to the control group, the group exercising at Zeitgeber Time 12 (ZT12), and the group exercising at ZT24. Control mice were not intervened, while ZT12 and ZT24 mice were trained for four weeks at the early and late time point of their active phase, respectively. We observed that the skeletal muscle of ZT12 mice exhibited higher total antioxidant capacity and lower reactive oxygen species compared to ZT24 mice. Furthermore, ZT12 mice improved the colocalization of BMAL1 with nucleus, the protein expression of BMAL1, NRF2, NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate-cysteine ligase modifier subunit and glutathione reductase in comparison to those of ZT24 mice. In conclusion, the 4-week aerobic training performed at ZT12 is more effective for enhancing NRF2-mediated antioxidant responses of skeletal muscle, which may be attributed to the specific activation of BMAL1.

IRF8 aggravates nonalcoholic fatty liver disease via BMAL1/PPARγ axis

Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic syndrome arising from lipid metabolic imbalance, with its prevalence increasing globally. In this study, we observed a significant up-regulation of interferon regulatory factor 8 (IRF8) in the liver of NAFLD model mice and patients. Overexpression of IRF8 induced lipid accumulation in the mouse primary hepatocytes. Mice with adeno-associated virus-mediated IRF8 overexpression exhibited hepatic steatosis due to up-regulated peroxisome proliferator-activated receptor γ (PPARγ) expression and increased fatty acid uptake and lipogenesis. In vitro, small interfering RNA-mediated IRF8 knockdown attenuated triglyceride accumulation by dampening PPARγ expression through transcriptional inhibition of brain and muscle ARNT-like 1. The PPARγ-specific antagonist GW9662 abolished the effect of IRF8 overexpression. Furthermore, adeno-associated virus-mediated IRF8 knockdown in the mouse liver markedly alleviated hepatic steatosis and obesity-related metabolic syndrome. These findings indicate that IRF8 plays a vital role in modulating hepatic lipid metabolism in a PPARγ-dependent manner and provide a previously unknown insight into NAFLD therapeutic strategies.

Potential Role of Bmal1 in Lipopolysaccharide-Induced Depression-Like Behavior and its Associated "Inflammatory Storm".

Inflammation plays an important role in the pathogenesis of depression; however, the underlying mechanisms remain unclear. Apart from the disordered circadian rhythm in animal models and patients with depression, dysfunction of clock genes has been reported to be involved with the progress of inflammation. This study aimed to investigate the role of circadian clock genes, especially brain and muscle ARNT-like 1 (Bmal1), in the linkage between inflammation and depression. Lipopolysaccharide (LPS)-challenged rats and BV2 cells were used in the present study. Four intraperitoneal LPS injections of 0.5mg/kg were administered once every other day to the rats, and BV2 cells were challenged with LPS for 24h at the working concentration of 1mg/L, with or without the suppression of Bmal1 via small interfering RNA. The results showed that LPS could successfully induce depression-like behaviors and an "inflammatory storm" in rats, as indicated by the increased immobility time in the forced swimming test and the decreased saccharin preference index in the saccharin preference test, together with hyperactivity of the hypothalamic-pituitary-adrenal axis, hyperactivation of astrocyte and microglia, and increased peripheral and central abundance of tumor necrosis factor-α, interleukin 6, and C-reactive protein. Moreover, the protein expression levels of brain-derived neurotrophic factor, triggering receptor expressed on myeloid cells 1, Copine6, and Synaptotagmin1 (Syt-1) decreased in the hippocampus and hypothalamus, whereas the expression of triggering receptor expressed on myeloid cells 2 increased. Interestingly, the fluctuation of temperature and serum concentration of melatonin and corticosterone was significantly different between the groups. Furthermore, protein expression levels of the circadian locomotor output cycles kaput, cryptochrome 2, and period 2 was significantly reduced in the hippocampus of LPS-challenged rats, whereas Bmal1 expression was significantly increased in the hippocampus but decreased in the hypothalamus, where it was co-located with neurons, microglia, and astrocytes. Consistently, apart from the reduced cell viability and increased phagocytic ability, LPS-challenged BV2 cells presented a similar trend with the changed protein expression in the hippocampus of the LPS model rats. However, the pathological changes in BV2 cells induced by LPS were reversed after the suppression of Bmal1. These results indicated that LPS could induce depression-like pathological changes, and the underlying mechanism might be partly associated with the imbalanced expression of Bmal1 and its regulated dysfunction of the circadian rhythm.

Cellular iron depletion enhances behavioral rhythm by limiting brain Per1 expression in mice.

Disturbances in the circadian rhythm are positively correlated with the processes of aging and related neurodegenerative diseases, which are also associated with brain iron accumulation. However, the role of brain iron in regulating the biological rhythm is poorly understood. In this study, we investigated the impact of brain iron levels on the spontaneous locomotor activity of mice with altered brain iron levels and further explored the potential mechanisms governing these effects invitro. Our results revealed that conditional knockout of ferroportin 1 (Fpn1) in cerebral microvascular endothelial cells led to brain iron deficiency, subsequently resulting in enhanced locomotor activity and increased expression of clock genes, including the circadian locomotor output cycles kaput protein (Clock) and brain and muscle ARNT-like 1 (Bmal1). Concomitantly, the levels of period circadian regulator 1 (PER1), which functions as a transcriptional repressor in regulating biological rhythm, were decreased. Conversely, the elevated brain iron levels in APP/PS1 mice inhibited autonomous rhythmic activity. Additionally, our findings demonstrate a significant decrease in serum melatonin levels in Fpn1cdh5 -CKO mice compared with the Fpn1flox/flox group. In contrast, APP/PS1 mice with brain iron deposition exhibited higher serum melatonin levels than the WT group. Furthermore, in the human glioma cell line, U251, we observed reduced PER1 expression upon iron limitation by deferoxamine (DFO; iron chelator) or endogenous overexpression of FPN1. When U251 cells were made iron-replete by supplementation with ferric ammonium citrate (FAC) or increased iron import through transferrin receptor 1 (TfR1) overexpression, PER1 protein levels were increased. Additionally, we obtained similar results to U251 cells in mouse cerebellar astrocytes (MA-c), where we collected cells at different time points to investigate the rhythmic expression of core clock genes and the impact of DFO or FAC treatment on PER1 protein levels. These findings collectively suggest that altered iron levels influence the circadian rhythm by regulating PER1 expression and thereby modulating the molecular circadian clock. In conclusion, our study identifies the regulation of brain iron levels as a potential new target for treating age-related disruptions in the circadian rhythm.

Differential Regulation of Circadian Clock Genes by UV-B Radiation and 1,25-Dihydroxyvitamin D: A Pilot Study during Different Stages of Skin Photocarcinogenesis.

Increasing evidence points at an important physiological role of the timekeeping system, known as the circadian clock (CC), regulating not only our sleep-awake rhythm but additionally many other cellular processes in peripheral tissues. It was shown in various cell types that environmental stressors, including ultraviolet B radiation (UV-B), modulate the expression of genes that regulate the CC (CCGs) and that these CCGs modulate susceptibility for UV-B-induced cellular damage. It was the aim of this pilot study to gain further insights into the CCs' putative role for UV-B-induced photocarcinogenesis of skin cancer. Applying RT-PCR, we analyzed the expression of two core CCGs (brain and muscle ARNT-like 1 (Bmal1) and Period-2 (Per2)) over several time points (0-60 h) in HaCaT cells with and without 1,25-dihydroxyvitamin D (D3) and/or UV-B and conducted a cosinor analysis to evaluate the effects of those conditions on the circadian rhythm and an extended mixed-effects linear modeling to account for both fixed effects of experimental conditions and random inter-individual variability. Next, we investigated the expression of these two genes in keratinocytes representing different stages of skin photocarcinogenesis, comparing normal (Normal Human Epidermal Keratinocytes-NHEK; p53 wild type), precancerous (HaCaT keratinocytes; mutated p53 status), and malignant (Squamous Cell Carcinoma SCL-1; p53 null status) keratinocytes after 12 h under the same conditions. We demonstrated that in HaCaT cells, Bmal1 showed a robust circadian rhythm, while the evidence for Per2 was limited. Overall expression of both genes, but especially for Bmal1, was increased following UV-B treatment, while Per2 showed a suppressed overall expression following D3. Both UVB and 1,25(OH)2D3 suggested a significant phase shift for Bmal1 (p < 0.05 for the acrophase), while no specific effect on the amplitude could be evidenced. Differential effects on the expression of BMAL1 and Per2 were found when we compared different treatment modalities (UV-B and/or D3) or cell types (NHEK, HaCaT, and SCL-1 cells). Comparing epidermal keratinocytes representing different stages of skin photocarcinogenesis, we provide further evidence for an independently operating timekeeping system in human skin, which is regulated by UV-B and disturbed during skin photocarcinogenesis. Our finding that this pattern of circadian rhythm was differentially altered by treatment with UV-B, as compared with treatment with D3, does not support the hypothesis that the expression of these CCGs may be regulated via UV-B-induced synthesis of vitamin D but might be introducing a novel photoprotective property of vitamin D through the circadian clock.

Repetitive Transcranial Magnetic Stimulation Alleviates MPTP-Induced Parkinson's Disease Symptoms by Regulating CaMKII-CREB-BMAL1 Pathway in Mice Model.

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulation technique that shows promise for the treatment of Parkinson's disease (PD). However, there is still limited understanding of the optimal stimulation frequencies and whether rTMS can alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. A PD mouse model was induced intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and treated with 1 Hz, 5 Hz, and 10 Hz rTMS. The neurological function, survival of dopaminergic neurons, and protein levels of Tyrosine hydroxylase (TH), α-synuclein(α-syn), and brain-derived neurotrophic factor (BDNF) in the striatum were measured to determine the optimal stimulation frequencies of rTMS treatment in PD mice. The levels of melatonin, cortisol, and the circadian rhythm of Brain and muscle ARNT-like 1 (BMAL1) in PD model mice were detected after optimal frequency rTMS treatment. Additionally, KN-93 and Bmal1siRNA interventions were used to verify that rTMS could alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. Administration of 10 Hz rTMS significantly improved neurological function, increased the protein levels of TH and BDNF, and inhibited abnormal aggregation of a-syn. Furthermore, administration of 10 Hz rTMS regulated the secretion profile of cortisol and melatonin and reversed the circadian arrhythmia of BMAL1 expression. After the KN-93 intervention, the MPTP+rTMS+KN-93 group exhibited decreased levels of P- Ca2+/calmodulin-dependent protein kinase II (CaMKII)/CaMKII, P-cAMP-response-element-binding protein (CREB)/CREB, BMALI, and TH. After Bmal1siRNA intervention, the protein levels of BMAL1 and TH were significantly reduced in the MPTP+10 Hz+ Bmal1siRNA group. At the same time, there were no significant changes in the proportions of P-CaMKIIα/CaMKIIα and P-CREB/CREB expression levels. Finally, immunohistochemical analysis showed that the number of TH-positive neurons was high in the MPTP+10 Hz group, but decreased significantly after KN-93 and Bmal1siRNA interventions. Treatment with 10 Hz rTMS alleviated MPTP-induced PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. This study provides a comprehensive perspective of the therapeutic mechanisms of rTMS in PD.

Beyond Conventional Therapies: Molecular Dynamics of Alzheimer's Treatment through CLOCK/BMAL1 Interactions.

Alzheimer's Disease (AD) represents a neurodegenerative disorder characterized by cognitive and behavioral impairments significantly hindering social and occupational functioning. Melatonin, a hormone pivotal in regulating the body's intrinsic circadian rhythm, also acts as a catalyst in the breakdown of beta-amyloid deposits, offering a promising therapeutic approach for AD. The upregulation of Brain and Muscle ARNT-Like 1 (Bmal1) gene expression, stimulated by melatonin, emerges as a potential contributor to AD intervention. Current pharmacological interventions, such as FDA-approved cholinesterase inhibitors and the recently authorized monoclonal antibody, Lecanemab, are utilized in AD management. However, the connection between these medications and Bmal1 remains insufficiently explored. This study aims to investigate the molecular effects of FDA-endorsed drugs on the CLOCK: Bmal1 dimer. Furthermore, considering the interactions between melatonin and Bmal1, this research explores the potential synergistic efficacy of combining these pharmaceutical agents with melatonin for AD treatment. Using molecular docking and MM/PBSA methodologies, this research determines the binding affinities of drugs within the Bmal1 binding site, constructing interaction profiles. The findings reveal that, among FDA-approved drugs, galanthamine and donepezil demonstrate notably similar binding energy values to melatonin, interacting within the Bmal1 binding site through analogous amino acid residues and functional groups. A novel therapeutic approach emerges, suggesting the combination of melatonin with Lecanemab as a monoclonal antibody therapy. Importantly, prior research has not explored the effects of FDA-approved drugs on Bmal1 expression or their potential for synergistic effects.

Artificial induction of circadian rhythm by combining exogenous BMAL1 expression and polycomb repressive complex 2 inhibition in human induced pluripotent stem cells.

Understanding the physiology of human-induced pluripotent stem cells (iPSCs) is necessary for directed differentiation, mimicking embryonic development, and regenerative medicine applications. Pluripotent stem cells (PSCs) exhibit unique abilities such as self-renewal and pluripotency, but they lack some functions that are associated with normal somatic cells. One such function is the circadian oscillation of clock genes; however, whether or not PSCs demonstrate this capability remains unclear. In this study, the reason why circadian rhythm does not oscillate in human iPSCs was examined. This phenomenon may be due to the transcriptional repression of clock genes resulting from the hypermethylation of histone H3 at lysine 27 (H3K27), or it may be due to the low levels of brain and muscle ARNT-like 1 (BMAL1) protein. Therefore, BMAL1-overexpressing cells were generated and pre-treated with GSK126, an inhibitor of enhancer of zest homologue 2 (EZH2), which is a methyltransferase of H3K27 and a component of polycomb repressive complex 2. Consequently, a significant circadian rhythm following endogenous BMAL1, period 2 (PER2), and other clock gene expression was induced by these two factors, suggesting a candidate mechanism for the lack of rhythmicity of clock gene expression in iPSCs.

Disruption of the Clock Component Bmal1 in Mice Promotes Cancer Metastasis through the PAI-1-TGF-β-myoCAF-Dependent Mechanism.

The circadian clock in animals and humans plays crucial roles in multiple physiological processes. Disruption of circadian homeostasis causes detrimental effects. Here, it is demonstrated that the disruption of the circadian rhythm by genetic deletion of mouse brain and muscle ARNT-like 1 (Bmal1) gene, coding for the key clock transcription factor, augments an exacerbated fibrotic phenotype in various tumors. Accretion of cancer-associated fibroblasts (CAFs), especially the alpha smooth muscle actin positive myoCAFs, accelerates tumor growth rates and metastatic potentials. Mechanistically, deletion of Bmal1 abrogates expression of its transcriptionally targeted plasminogen activator inhibitor-1 (PAI-1). Consequently, decreased levels of PAI-1 in the tumor microenvironment instigate plasmin activation through upregulation of tissue plasminogen activator and urokinase plasminogen activator. The activated plasmin converts latent TGF-β into its activated form, which potently induces tumor fibrosis and the transition of CAFs into myoCAFs, the latter promoting cancer metastasis. Pharmacological inhibition of the TGF-β signaling largely ablates the metastatic potentials of colorectal cancer, pancreatic ductal adenocarcinoma, and hepatocellular carcinoma. Together, these data provide novel mechanistic insights into disruption of the circadian clock in tumor growth and metastasis. It is reasonably speculated that normalization of the circadian rhythm in patients provides a novel paradigm for cancer therapy.

Circadian clock protein Bmal1 accelerates acute myeloid leukemia by inhibiting ferroptosis through the EBF3/ALOX15 axis.

Acute myeloid leukemia (AML) is a major leukemia with high mortality. Ferroptosis is an important regulator of cancers. However, the role of ferroptosis and its regulatory mechanisms in AML remain largely unknown. In this study, we reported elevated brain and muscle ARNT-Like protein-1 (Bmal1) expression in AML patients and cell lines, and its upregulation indicated the poor survival of patients. The correlation analysis showed that Bmal1 expression was closely correlated with cytogenetics and the French-American-British subtypes, but was not correlated with age, gender and white blood cells. RSL3 reduced Bmal1 expression in HL-60 and NB4 cells. Malondialdehyde, total iron, Fe2+ , glutathione and lipid peroxidation were examined to evaluate ferroptosis. Overexpression of Bmal1 repressed RSL3-induced ferroptosis in AML cells. Bmal1 recruited Enhancer of zeste homolog 2 (EZH2) to the Early B cell factor 3 (EBF3) promoter and enhanced its methylation, thus suppressing EBF3 expression. Moreover, the knockdown of Bmal1 sensitized AML cells to RSL3-induced ferroptosis, and it was counteracted by EBF3 knockdown. Furthermore, EBF3 bound to the Arachidonate 15-pipoxygenase (ALOX15) promoter to enhance its expression, and overexpression of EBF3 enhanced RSL3-induced ferroptosis dependent on ALOX5. We established a subcutaneous AML xenograft tumor model and reported that knockdown of Bmal1 and overexpression of EBF3 restrained AML growth by promoting ALOX15-mediated ferroptosis in vivo. Collectively, Bmal1 inhibits RSL3-induced ferroptosis by promoting EZH2-mediated EBF3 methylation and suppressing the expression of EBF3 and ALOX15, thus accelerating AML.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein RP58 is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin synthesis pathway (TPH1, ASMT and AANAT) as well as clock genes (CLOCK and BMAL1), and it also reduced the production of melatonin, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of melatonin synthesis enzymes (TPH1, ASMT and AANAT) and clock genes (CLOCK and BMAL1) and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effects of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous melatonin. Furthermore, exogenous melatonin reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in melatonin production, voluntary activity periods, and activity frequency as well as a diminished decrease in the expression of melatonin synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI. This article is protected by copyright. All rights reserved.

Exposure to polychlorinated biphenyls selectively dysregulates endothelial circadian clock and endothelial toxicity

Polychlorinated biphenyls (PCBs) are lipophilic and persistent environmental toxicants, which pose health threats to the exposed population. Among several organs and cell types, vascular tissue and endothelial cells are especially prone to PCB-induced toxicity. Exposure to PCBs can exert detrimental impacts on biological pathways, expression of transcription factors, and tight junction proteins that are integral to the functionality of endothelial cells. Because biological and cellular processes are tightly regulated by circadian rhythms, and disruption of the circadian system may cause several diseases, we evaluated if exposure to PCBs can alter the expression of the major endothelial circadian regulators. In addition, we studied if dysregulation of circadian rhythms by silencing the brain and muscle ARNT-like 1 (Bmal1) gene can contribute to alterations of brain endothelial cells in response to PCB treatment. We demonstrated that diminished expression of Bmal1 enhances PCB-induced dysregulation of tight junction complexes, such as the expression of occludin, JAM-2, ZO-1, and ZO-2 especially at pathologically relevant longer PCB exposure times. Overall, the obtained results imply that dysregulation of the circadian clock is involved in endothelial toxicity of PCBs. The findings provide new insights for toxicological studies focused on the interactions between environmental pollutants and regulation of circadian rhythms.

BMAL1 regulates MUC1 overexpression in ovalbumin-induced asthma.

Asthma often presents with a daily rhythm; however, the underlying mechanisms remain unclear. Circadian rhythm genes have been proposed to regulate inflammation and mucin expression. Here, ovalbumin (OVA)-induced mice and serum shock human bronchial epidermal cells (16HBE) were used in in vivo and in vitro models, respectively. We constructed a brain and muscle ARNT-like 1 (BMAL1) knockdown 16HBE cell line to analyze the effects of rhythmic fluctuations on mucin expression. Serum immunoglobulin E (IgE) and circadian rhythm genes in asthmatic mice showed rhythmic fluctuation amplitude. Mucin (MUC) 1 and MUC5AC expression was increased in the lung tissue of the asthmatic mice. MUC1 expression was negatively correlated with that of the circadian rhythm genes, particularly BMAL1 (r=-0.546, P=0.006). There was also a negative correlation between BMAL1 and MUC1 expression (r=-0.507, P=0.002) in the serum shock 16HBE cells. BMAL1 knockdown negated the rhythmic fluctuation amplitude of MUC1 expression and upregulated MUC1 expression in the 16HBE cells. These results indicate that the key circadian rhythm gene, BMAL1, causes periodic changes in airway MUC1 expression in OVA-induced asthmatic mice. Targeting BMAL1 to regulate periodic changes in MUC1 expression may, therefore, improve asthma treatments.

BMAL1 Promotes Valvular Interstitial Cells' Osteogenic Differentiation through NF-κ B/AKT/MAPK Pathway.

Calcific aortic valve disease (CAVD) is most common in the aging population and is without effective medical treatments. Brain and muscle ARNT-like 1 (BMAL1) is related to calcification. It has unique tissue-specific characteristics and plays different roles in different tissues' calcification processes. The purpose of the present study is to explore the role of BMAL1 in CAVD. The protein levels of BMAL1 in normal and calcified human aortic valves and valvular interstitial cells (VICs) isolated from normal and calcified human aortic valves were checked. HVICs were cultured in osteogenic medium as an in vitro model, and BMAL1 expression and location were detected. TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA were applied to detect the mechanism underlying the source of BMAL1 during HVICs' osteogenic differentiation. ChIP was applied to check whether BMAL1 could directly interact with the runx2 primer CPG region, and the expression of key proteins involved in the TNF signaling pathway and NF-κ B pathway was tested after silencing BMAL1. In this study, we found that BMAL1 expression was elevated in calcified human aortic valves and VICs isolated from calcified human aortic valves. Osteogenic medium could promote BMAL1 expression in HVICs and the knockdown of BMAL1 induced the inhibition of HVICs' osteogenic differentiation. Furthermore, the osteogenic medium promoting BMAL1 expression could be blocked by TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA. Meanwhile, BMAL1 could not bind with the runx2 primer CPG region directly, but knockdown of BMAL1 led to decreased levels of P-AKT, P-IκBα, P-p65 and P-JNK. Osteogenic medium could promote BMAL1 expression in HVICs through the TGF-β/RhoA/ROCK pathway. BMAL1 could not act as a transcription factor, but functioned through the NF-κ B/AKT/MAPK pathway to regulate the osteogenic differentiation of HVICs.

Circadian Rhythm Regulator REV-ERBα Attenuates Neuroapoptosis in Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

Subarachnoid hemorrhage (SAH) is associated with circadian rhythm abnormalities, in which REV-ERBα plays a major regulatory role. Our ambition was to investigate the capacity of REV-ERBα to inhibit neuronal neuroapoptosis induced by early brain injury (EBI) after SAH. The endovascular perforation model was used to produce experimental SAH in Sprague-Dawley rats. Specific small-interfering RNA was used to downregulate the expression REV-ERBα while SR9009 was used to upregulate the expression before assessments. Short- and long-term neurobehavior assessments, immunofluorescence staining, TUNEL staining, Nissl staining, brain water content, and Western blot were performed. The expression level of endogenous REVERBα tended to increase and then decrease after SAH and peaked at 48h. REV-ERBα upregulation diminished neuronal apoptosis and enhanced neurological function deficits. Meanwhile, REV-ERBα downregulation aggravated the damage. Furthermore, the levels of downstream proteins of REV-ERBα (i.e., brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK)) changed accordingly with REV-ERBα regulation. REV-ERBα may attenuate neuronal apoptosis in EBI after SAH through the BMAL1/CLOCK pathway.