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Redox Biology Aug 2023Brain and muscle arnt-like protein 1 (Bmal1) is a crucial transcription factor, regulating circadian rhythm and involved in multiple heart diseases. However, it is...
Brain and muscle arnt-like protein 1 (Bmal1) is a crucial transcription factor, regulating circadian rhythm and involved in multiple heart diseases. However, it is unknown whether Bmal1 promotes diabetic cardiomyopathy (DCM) pathogenesis. The objective of this investigation was to ascertain the vital role of Bmal1 in the progression of DCM. Mice with T2D and H9c2 cardiomyoblasts exposed to high glucose and palmitic acid (HGHP) were used. Cardiomyocyte-specific knockout mouse of Bmal1 (CKB) was also generated, and cardiac Bmal1 was overexpressed in type 2 diabetes (T2D) mice using an adeno-associated virus. Bmal1 gene recombinant adenovirus was used to either knockdown or overexpress in H9c2 cardiomyoblasts. Bmal1 expression was significantly altered in diabetic mice hearts. Bmal1 downregulation in CKB and T2D mice heart accelerated cardiac hypertrophy and diastolic dysfunction, while Bmal1 overexpression ameliorated these pathological changes in DCM mice. Furthermore, DCM mice had significant mitochondrial ultrastructural defects, reactive oxygen species accumulation, and apoptosis, which could be alleviated by overexpressing Bmal1. In H9c2 cardiomyoblasts, genetic downregulation of Bmal1 or HGHP markedly decreased the binding of Bcl2 to IP3R, thus increasing Ca release to mitochondria through mitochondria-associated endoplasmic reticulum membranes. Importantly, chromatin immunoprecipitation revealed Bmal1 could bind directly to the Bcl2 gene promoter region. Bmal1 overexpression augmented the Bmal1/Bcl2 binding, enhancing the inhibition of Bcl2 on IP3R activity, thus alleviating mitochondrial Ca overload and subsequent cell apoptosis. These results show that Bmal1 is involved in the DCM development through Bcl2/IP3R-mediated mitochondria Ca overload. Therapy targeting the circadian clock (Bmal1) can treat DCM.
Topics: Animals; Mice; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Down-Regulation; Mice, Knockout; Mitochondria
PubMed: 37356134
DOI: 10.1016/j.redox.2023.102788 -
Clinical Cancer Research : An Official... Nov 2023Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy.
PURPOSE
Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy.
EXPERIMENTAL DESIGN
Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess ERBB2 and CCNE1 expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent in situ hybridization, and IHC. In vitro, CCNE1 was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. In vivo, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array.
RESULTS
Among several ERBB2-amplified cancers, CCNE1 co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd). In vitro, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. In vivo, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd-treated colon cancer model.
CONCLUSIONS
We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring CCNE1 amplifications. See related commentary by Rolfo et al., p. 4317.
Topics: Humans; Animals; Mice; Cyclin E; In Situ Hybridization, Fluorescence; Trastuzumab; Receptor, ErbB-2; Camptothecin; Immunoconjugates; Neoplasms
PubMed: 37279095
DOI: 10.1158/1078-0432.CCR-23-0103 -
Annals of Medicine Dec 2023Excessive oxygen free radicals and toxic substances are generated in cerebral ischemia-reperfusion (I/R) process. Dexmedetomidine (DEX), a common anesthetic and sedative...
Excessive oxygen free radicals and toxic substances are generated in cerebral ischemia-reperfusion (I/R) process. Dexmedetomidine (DEX), a common anesthetic and sedative drug, can considerably boost glutathione (GSH), which has anti-copper influx effects. Focusing on cuproptosis, the mechanism of DEX in the I/R was revealed. Using the I/R rat model, the effects of DEX and the copper chelator D-penicillamine on cerebral infarct volume, copper levels, mitochondrial respiration and membrane potential, GSH content, and enrichment of cuproptosis functional proteins were examined. The involvement of ferredoxin 1 (FDX1) in the DEX regulatory pathway was verified by overexpressing FDX1 . DEX could significantly reduce cerebral infarction in rats, reduce copper levels, maintain mitochondrial functions, increase GSH, and reduce the content of key proteins related to cuproptosis. These aspects were replicated and revealed that FDX1 overexpression partially reversed the impacts of DEX. Together, cuproptosis occurs in the brain I/R process and DEX can enhance cell survival by blocking the primary pathway mediated by FDX1.KEY MESSAGESDexmedetomidine reduces cerebral infarction in the I/R rat models.Dexmedetomidine reduces cuproptosis in the I/R rat models.FDX1, an upstream of protein fatty acylation, mediates regulation of Dexmedetomidine.
Topics: Animals; Rats; Apoptosis; Brain Ischemia; Cerebral Infarction; Dexmedetomidine; Ferredoxins; Homeostasis; Reperfusion; Reperfusion Injury
PubMed: 37162502
DOI: 10.1080/07853890.2023.2209735 -
Cell Death & Disease Jul 2023Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs)...
Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs) constitute a family of C2/H2 zinc finger DNA-binding proteins. KLF6, a member of the KLF protein family, plays crucial roles in proliferation, metabolism, inflammation, and injury responses; however, its role in HIR is largely remains unknown. After I/R injury, we found that KLF6 expression in mice and hepatocytes was significantly upregulated. Mice were then subjected to I/R following injection of shKLF6- and KLF6-overexpressing adenovirus through the tail vein. KLF6 deficiency markedly exacerbated liver damage, cell apoptosis, and activation of hepatic inflammatory responses, whereas hepatic overexpression of KLF6 in mice produced the opposite results. In addition, we knocked out or overexpressed KLF6 in AML12 cells before exposing them to a hypoxia-reoxygenation challenge. KLF6 knockout decreased cell viability and increased hepatocyte inflammation, apoptosis, and ROS, whereas KLF6 overexpression had the opposite effects. Mechanistically, KLF6 inhibited the overactivation of autophagy at the initial stage, and the regulatory effect of KLF6 on I/R injury was autophagy-dependent. CHIP-qPCR and luciferase reporter gene assays confirmed that KLF6 bound to the promoter region of Beclin1 and inhibited its transcription. Additionally, KLF6 activated the mTOR/ULK1 pathway. Finally, we performed a retrospective analysis of the clinical data of liver transplantation patients and identified significant associations between KLF6 expression and liver function following liver transplantation. In conclusion, KLF6 inhibited the overactivation of autophagy via transcriptional regulation of Beclin1 and activation of the mTOR/ULK1 pathway, thereby protecting the liver from I/R injury. KLF6 is expected to serve as a biomarker for estimating the severity of I/R injury following liver transplantation.
Topics: Animals; Mice; Autophagy; Beclin-1; Inflammation; Liver; Retrospective Studies; Kruppel-Like Factor 6
PubMed: 37391422
DOI: 10.1038/s41419-023-05872-3 -
Plant Communications May 2024In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat...
In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.
Topics: Triticum; Transformation, Genetic; Plant Proteins; Plants, Genetically Modified; Regeneration; Gene Expression Regulation, Plant
PubMed: 37897039
DOI: 10.1016/j.xplc.2023.100738 -
Redox Biology Sep 2023Vascular endothelial cells (ECs) senescence plays a crucial role in vascular aging that promotes the initiation and progression of cardiovascular disease. The mutation...
Vascular endothelial cells (ECs) senescence plays a crucial role in vascular aging that promotes the initiation and progression of cardiovascular disease. The mutation of Grb10-interacting GYF protein 2 (GIGYF2) is strongly associated with the pathogenesis of aging-related diseases, whereas its role in regulating ECs senescence and dysfunction still remains elusive. In this study, we found aberrant hyperexpression of GIGYF2 in senescent human ECs and aortas of old mice. Silencing GIGYF2 in senescent ECs suppressed eNOS-uncoupling, senescence, and endothelial dysfunction. Conversely, in nonsenescent cells, overexpressing GIGYF2 promoted eNOS-uncoupling, cellular senescence, endothelial dysfunction, and activation of the mTORC1-SK61 pathway, which were ablated by rapamycin or antioxidant N-Acetyl-l-cysteine (NAC). Transcriptome analysis revealed that staufen double-stranded RNA binding protein 1 (STAU1) is remarkably downregulated in the GIGYF2-depleted ECs. STAU1 depletion significantly attenuated GIGYF2-induced cellular senescence, dysfunction, and inflammation in young ECs. Furthermore, we disclosed that GIGYF2 acting as an RNA binding protein (RBP) enhances STAU1 mRNA stability, and that the intron region of the late endosomal/lysosomal adaptor MAPK and mTOR activator 4 (LAMTOR4) could bind to STAU1 protein to upregulate LAMTOR4 expression. Immunofluorescence staining showed that GIGYF2 overexpression promoted the translocation of mTORC1 to lysosome. In the mice model, GIGYF2 Cdh-Cre mice protected aged mice from aging-associated vascular endothelium-dependent relaxation and arterial stiffness. Our work discloses that GIGYF2 serving as an RBP enhances the mRNA stability of STAU1 that upregulates LAMTOR4 expression through binding with its intron region, which activates the mTORC1-S6K1 signaling via recruitment of mTORC1 to the lysosomal membrane, ultimately leading to ECs senescence, dysfunction, and vascular aging. Disrupting the GIGYF2-STAU1-mTORC1 signaling cascade may represent a promising therapeutic approach against vascular aging and aging-related cardiovascular diseases.
Topics: Animals; Humans; Mice; Aging; Carrier Proteins; Cellular Senescence; Cytoskeletal Proteins; Endothelial Cells; Guanine Nucleotide Exchange Factors; Mechanistic Target of Rapamycin Complex 1; RNA-Binding Proteins
PubMed: 37517320
DOI: 10.1016/j.redox.2023.102824 -
The European Respiratory Journal Jul 2023In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and...
BACKGROUND
In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise.
METHODS
We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following overexpression in collagen-producing cells.
RESULTS
TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log fold change (FC) 8.909, adjusted p-value 1.82×10) and control fibroblasts (logFC 8.975, adjusted p-value 3.72×10). expression was selectively upregulated in IPF myofibroblasts (logFC 3.136, adjusted p-value 1.41×10 ), with two regions of having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts.
CONCLUSIONS
Our studies utilising human multiomic single-cell analyses combined with murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.
Topics: Humans; Mice; Animals; Myofibroblasts; Chromatin; Idiopathic Pulmonary Fibrosis; Lung; Fibroblasts; Collagen; Fibrosis; Bleomycin; Transcription Factors; RNA; Nuclear Proteins; Twist-Related Protein 1
PubMed: 37142338
DOI: 10.1183/13993003.00474-2022 -
Nature Feb 2024Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly...
Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors. Single-cell transcriptomics and comparison to independent neural stem cells showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.
Topics: Humans; Amyotrophic Lateral Sclerosis; C-Reactive Protein; DNA-Binding Proteins; Frontotemporal Lobar Degeneration; Nerve Net; Nerve Tissue Proteins; Neural Stem Cells; Neuroglia; Neurons; Reproducibility of Results
PubMed: 38355792
DOI: 10.1038/s41586-024-07042-7 -
Signal Transduction and Targeted Therapy Oct 2023Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the predominant impairment of neurons in the hippocampus and the formation of amyloid plaques,...
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the predominant impairment of neurons in the hippocampus and the formation of amyloid plaques, hyperphosphorylated tau protein, and neurofibrillary tangles in the brain. The overexpression of amyloid-β precursor protein (APP) in an AD brain results in the binding of APP intracellular domain (AICD) to Fe65 protein via the C-terminal Fe65-PTB2 interaction, which then triggers the secretion of amyloid-β and the consequent pathogenesis of AD. Apparently, targeting the interaction between APP and Fe65 can offer a promising therapeutic approach for AD. Recently, exosome, a type of extracellular vesicle with diameter around 30-200 nm, has gained much attention as a potential delivery tool for brain diseases, including AD, due to their ability to cross the blood-brain barrier, their efficient uptake by autologous cells, and their ability to be surface-modified with target-specific receptor ligands. Here, the engineering of hippocampus neuron cell-derived exosomes to overexpress Fe65, enabled the development of a novel exosome-based targeted drug delivery system, which carried Corynoxine-B (Cory-B, an autophagy inducer) to the APP overexpressed-neuron cells in the brain of AD mice. The Fe65-engineered HT22 hippocampus neuron cell-derived exosomes (Fe65-EXO) loaded with Cory-B (Fe65-EXO-Cory-B) hijacked the signaling and blocked the natural interaction between Fe65 and APP, enabling APP-targeted delivery of Cory-B. Notably, Fe65-EXO-Cory-B induced autophagy in APP-expressing neuronal cells, leading to amelioration of the cognitive decline and pathogenesis in AD mice, demonstrating the potential of Fe65-EXO-Cory-B as an effective therapeutic intervention for AD.
Topics: Mice; Animals; Alzheimer Disease; Exosomes; Nerve Tissue Proteins; Amyloid beta-Protein Precursor; Cognition; Neurons
PubMed: 37867176
DOI: 10.1038/s41392-023-01657-4 -
Journal of Nanobiotechnology Aug 2023Stabilization and increased activity of hypoxia-inducible factor 1-α (HIF-1α) can directly increase cancellous bone formation and play an essential role in bone...
BACKGROUND
Stabilization and increased activity of hypoxia-inducible factor 1-α (HIF-1α) can directly increase cancellous bone formation and play an essential role in bone modeling and remodeling. However, whether an increased HIF-1α expression in adipose-derived stem cells (ADSCs) increases osteogenic capacity and promotes bone regeneration is not known.
RESULTS
In this study, ADSCs transfected with small interfering RNA and HIF-1α overexpression plasmid were established to investigate the proliferation, migration, adhesion, and osteogenic capacity of ADSCs and the angiogenic ability of human umbilical vein endothelial cells (HUVECs). Overexpression of HIF-1α could promote the biological functions of ADSCs, and the angiogenic ability of HUVECs. Western blotting showed that the protein levels of osteogenesis-related factors were increased when HIF-1α was overexpressed. Furthermore, the influence of upregulation of HIF-1α in ADSC sheets on osseointegration was evaluated using a Sprague-Dawley (SD) rats implant model, in which the bone mass and osteoid mineralization speed were evaluated by radiological and histological analysis. The overexpression of HIF-1α in ADSCs enhanced bone remodeling and osseointegration around titanium implants. However, transfecting the small interfering RNA (siRNA) of HIF-1α in ADSCs attenuated their osteogenic and angiogenic capacity. Finally, it was confirmed in vitro that HIF-1α promotes osteogenic differentiation and the biological functions in ADSCs via the VEGF/AKT/mTOR pathway.
CONCLUSIONS
This study demonstrates that HIF-1α has a critical ability to promote osteogenic differentiation in ADSCs by coupling osteogenesis and angiogenesis via the VEGF/AKT/mTOR signaling pathway, which in turn increases osteointegration and bone formation around titanium implants.
Topics: Animals; Humans; Rats; Human Umbilical Vein Endothelial Cells; Neovascularization, Physiologic; Osteogenesis; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Titanium; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Hypoxia-Inducible Factor 1, alpha Subunit
PubMed: 37550736
DOI: 10.1186/s12951-023-02020-z