RESUMO
MIR125B, particularly its 5p strand, is apparently involved in multiple cellular processes, including osteoblastogenesis and osteoclastogenesis. Given that MIR125B is transcribed from the loci Mir125b1 and Mir125b2, three mature transcripts (MIR125B-5p, MIR125B1-3p, and MIR125B2-3p) are generated (MIR125B-5p is common to both); however, their expression profiles and roles in the bones remain poorly understood. Both primary and mature MIR125B transcripts were differentially expressed in various organs, tissues, and cells, and their expression patterns did not necessarily correlate in wild-type (WT) mice. We generated Mir125b2 knockout (KO) mice to examine the contribution of Mir125b2 to MIR125B expression profiles and bone phenotypes. Mir125b2 KO mice were born and grew normally without any changes in bone parameters. Interestingly, in WT and Mir125b2 KO, MIR125B-5p was abundant in the calvaria and bone marrow stromal cells. These results indicate that the genetic ablation of Mir125b2 does not impinge on the bones of mice, attracting greater attention to MIR125B-5p derived from Mir125b1. Future studies should investigate the conditional deletion of Mir125b1 and both Mir125b1 and Mir125b2 in mice.
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Osso e Ossos , Camundongos Knockout , MicroRNAs , Fenótipo , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Osso e Ossos/metabolismo , Osteogênese/genética , Camundongos Endogâmicos C57BL , Crânio/metabolismoRESUMO
Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.
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Camundongos Knockout , Contração Muscular , Músculo Esquelético , Fosfatidilcolinas , Animais , Músculo Esquelético/metabolismo , Camundongos , Fosfatidilcolinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Diester Fosfórico HidrolasesRESUMO
Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.
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DNA Polimerase Dirigida por DNA , Metilnitrosoureia , Mutagênese , Nucleotidiltransferases , Neoplasias do Timo , Animais , Camundongos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Sequenciamento do Exoma , Linfoma/genética , Linfoma/induzido quimicamente , Linfoma/patologia , Metilnitrosoureia/toxicidade , Camundongos Transgênicos , Mutação , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Neoplasias do Timo/genética , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/patologiaRESUMO
Apolipoprotein E4 (APOE4), the strongest risk factor for late-onset Alzheimer's disease (AD), has been revealed to cause greater accumulation of extracellular amyloid ß (Aß) aggregates than does APOE3 in traditional transgenic mouse models of AD. However, concerns that the overexpression paradigm might have affected the phenotype remain. Amyloid precursor protein (APP)-knock-in (KI) mice, incorporating APP mutations associated with AD development, offer an alternative approach for overproducing pathogenic Aß without needing overexpression of APP. Here, we present the results of comprehensive analyses of pathological and biochemical traits in the brains of APP-KI mice harboring APP-associated familial AD mutations (APPNL-G-F/NL-G-F mice) crossed with human APOE-KI mice. Immunohistochemical and biochemical analyses revealed the APOE genotype-dependent increase in Aß pathology and glial activation, which was evident within 8 months in the mouse model. These results suggested that this mouse model may be valuable for investigating APOE pathobiology within a reasonable experimental time frame. Thus, this model can be considered in investigating the interaction between APOE and Aß in vivo, which may not be addressed appropriately by using other transgenic mouse models.
Assuntos
Doença de Alzheimer , Camundongos , Humanos , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/genética , Camundongos Transgênicos , Apolipoproteína E3/genética , Genótipo , Modelos Animais de DoençasRESUMO
DNA damage contributes to atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be established. Here, we investigated the role of DSBs in atherosclerosis using mice and vascular cells deficient in Ku80, a DSB repair protein. After 4 weeks of a high-fat diet, Ku80-deficient apolipoprotein E knockout mice (Ku80+/-ApoE-/-) displayed increased plaque size and DSBs in the aorta compared to those of ApoE-/- control. In the preatherosclerotic stages (two-week high-fat diet), the plaque size was similar in both the Ku80+/-ApoE-/- and ApoE-/- control mice, but the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80+/-ApoE-/- aortas. We further investigated molecular links between DSBs and inflammatory responses using vascular smooth muscle cells isolated from Ku80 wild-type and Ku80+/- mice. The Ku80+/- cells displayed senescent features and elevated levels of inflammatory cytokine mRNAs. Moreover, the cytosolic DNA-sensing cGAS-STING pathway was activated in the Ku80+/- cells. Inhibiting the cGAS-STING pathway reduced IL-6 mRNA level. Notably, interferon regulatory factor 3 (IRF3), a downstream effector of the cGAS-STING pathway, was activated, and the depletion of IRF3 also reduced IL-6 mRNA levels in the Ku80+/- cells. Finally, DSBs accumulation in normal cells also activated the cGAS-STING-IRF3 pathway. In addition, cGAS inhibition attenuated DNA damage-induced IL-6 expression and cellular senescence in these cells. These results suggest that DSBs accumulation promoted atherosclerosis by upregulating proinflammatory responses and cellular senescence via the cGAS-STING (-IRF3) pathway.
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Aterosclerose , Quebras de DNA de Cadeia Dupla , Placa Aterosclerótica , Animais , Camundongos , Apolipoproteínas E , Aterosclerose/genética , Citocinas/metabolismo , DNA/metabolismo , Interleucina-6 , Camundongos Knockout , Nucleotidiltransferases/metabolismoRESUMO
BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.
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Esclerose Lateral Amiotrófica , Proteínas de Ciclo Celular , Proteínas de Membrana Transportadoras , Viroses , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Proteínas de Ciclo Celular/genética , Imunidade Inata , Interferon beta/genética , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos KnockoutRESUMO
A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3'-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.
Assuntos
MicroRNAs , NF-kappa B , Animais , Camundongos , Inflamação/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de SinaisRESUMO
Age at exposure is a major modifier of radiation-induced carcinogenesis. We used mouse models to elucidate the mechanism underlying age-related susceptibility to radiation-induced tumorigenesis. Radiation exposure in infants was effective at inducing tumors in B6/B6-Chr18MSM-F1 ApcMin/+ mice. Loss of heterozygosity analysis revealed that interstitial deletion may be considered a radiation signature in this model and tumor number containing a deletion correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. Furthermore, in Lgr5-eGFP-ires-CreERT2; Apcflox/flox mice, deletions of both floxed Apc alleles in Lgr5-positive stem cells in infants resulted in the formation of more tumors than in adults. These results suggest that tumorigenicity of Apc-deficient stem cells varies with age and is higher in infant mice. Three-dimensional immunostaining analyses indicated that the crypt architecture in the intestine of infants was immature and different from that in adults concerning crypt size and the number of stem cells and Paneth cells per crypt. Interestingly, the frequency of crypt fission correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. During crypt fission, the percentage of crypts with lysozyme-positive mature Paneth cells was lower in infants than that in adults, whereas no difference in the behavior of stem cells or Paneth cells was observed regardless of age. These data suggest that morphological dynamics in intestinal crypts affect age-dependent susceptibility to radiation-induced tumorigenesis; oncogenic mutations in infant stem cells resulting from radiation exposure may acquire an increased proliferative potential for tumor induction compared with that in adults.
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Intestinos , Células-Tronco , Camundongos , Animais , Intestinos/patologia , Células-Tronco/patologia , Carcinogênese/genética , Carcinogênese/patologia , Mucosa IntestinalRESUMO
We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.
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Neuroblastoma , Vitamina A , Linhagem Celular , Humanos , Neuroblastoma/patologia , Ácido Retinoico 4 Hidroxilase/genética , Tetraciclinas , TransfecçãoRESUMO
Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.
Assuntos
Glaucoma , Traumatismos do Nervo Óptico , Animais , Axônios/patologia , Glaucoma/metabolismo , Camundongos , Camundongos Knockout , Compressão Nervosa , Regeneração Nervosa/fisiologia , Nervo Óptico , Traumatismos do Nervo Óptico/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Ganglionares da Retina/metabolismo , Zimosan/metabolismo , Zimosan/farmacologiaRESUMO
The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-ß2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-ß2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.
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In the male germline, the machinery to repress retrotransposons that threaten genomic integrity via the piRNA pathway is established in gonocytes. It has been reported that disruption of the piRNA pathway leads to activation of retrotransposons and arrests spermatogenesis before it enters the second meiosis; however, its effects on gonocytes have not been fully elucidated. In this study, we analyzed the effects of Asz1 deletion, which is a crucial component of the piRNA pathway, on the gonocyte transcriptome. In Asz1-null gonocytes, MIWI2, which is responsible for introducing DNA methylation to retrotransposons in a piRNA-dependent manner, disappeared from the nuclei of fetal gonocytes. Transcriptome analysis revealed that retrotransposons targeted by the piRNA pathway and non-annotated transcript variants were upregulated in gonocytes from neonatal Asz1-/- mice. These non-annotated transcript variants were chimeras generated by joining exons transcribed from retrotransposons and canonical genes. DNA methylation analysis showed that retrotransposons that induce the expression of aberrant chimeric transcripts are not fully methylated. This was consistent with the impaired nuclear localization of MIWI2 in Asz1-null gonocytes. Furthermore, heterogeneity of DNA methylation status in retrotransposons was observed in both gonocytes and their descendants. This suggests that the piRNA system in gonocytes can potentially prevent spermatogenic cell populations bearing aberrant chimeric transcripts from propagating later in spermatogenesis. In conclusion, Asz1 is required to repress retrotransposons and retrotransposon-driven aberrant chimeric transcripts in gonocytes through the piRNA pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Células Germinativas , Espermatogênese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Argonautas/genética , Quimera , Deleção de Genes , Células Germinativas/metabolismo , Masculino , Camundongos , RNA Interferente Pequeno/metabolismo , Retroelementos , Espermatogênese/genética , Testículo/metabolismoRESUMO
We recently reported an unexpected role of osteoblast-derived matrix vesicles in the delivery of microRNAs to bone matrix. Of such microRNAs, we found that miR-125b inhibited osteoclast formation by targeting Prdm1 encoding a transcriptional repressor of anti-osteoclastogenesis factors. Transgenic (Tg) mice overexpressing miR-125b in osteoblasts by using human osteocalcin promoter grow normally but exhibit high trabecular bone mass. We have now further investigated the effects of osteoblast-mediated miR-125b overexpression on skeletal morphogenesis and remodeling during development, aging and in a situation of skeletal repair, i.e., fracture healing. There were no significant differences in the growth plate, primary spongiosa or lateral (periosteal) bone formation and mineral apposition rate between Tg and wild-type (WT) mice during early bone development. However, osteoclast number and medial (endosteal) bone resorption were less in Tg compared to WT mice, concomitant with increased trabecular bone mass. Tg mice were less susceptible to age-dependent changes in bone mass, phosphate/amide I ratio and mechanical strength. In a femoral fracture model, callus formation progressed similarly in Tg and WT mice, but callus resorption was delayed, reflecting the decreased osteoclast numbers associated with the Tg callus. These results indicate that the decreased osteoclastogenesis mediated by miR-125b overexpression in osteoblasts leads to increased bone mass and strength, while preserving bone formation and quality. They also suggest that, in spite of the fact that single miRNAs may target multiple genes, the miR-125b axis may be an attractive therapeutic target for bone loss in various age groups.
Assuntos
Desenvolvimento Ósseo , Reabsorção Óssea/patologia , MicroRNAs/genética , Osteoblastos/patologia , Osteoclastos/patologia , Osteogênese , Fatores Etários , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismoRESUMO
The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus-positive (Venus+) cells expressing the fluorescent protein Venus under the control of the 2.3-kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single-cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus+ single cells indicated that Venus+ cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster-specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co-expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone-forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Genetically modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Here we present a fully automated system for microinjection into the pronucleus to facilitate genetic modification. We first developed software that automatically controls the microinjection system hardware. The software permits automatic rotation of the zygote to move the pronucleus to the injection pipette insertion position. We also developed software that recognizes the pronucleus in 3-dimensional coordinates so that the injection pipette can be automatically inserted into the pronucleus, and achieved a 94% insertion rate by linking the 2 pieces of software. Next, we determined the optimal solution injection conditions (30 hPa, 0.8-2.0 s) by examining the survival rate of injected zygotes. Finally, we produced transgenic (traditional DNA injection and piggyBac Transposon system) and knock-in (genomic editing) mice using our newly developed Integrated Automated Embryo Manipulation System (IAEMS). We propose that the IAEMS will simplify highly reproducible pronuclear stage zygote microinjection procedures.
Assuntos
Pesquisas com Embriões , Embrião de Mamíferos , Engenharia Genética/métodos , Camundongos Transgênicos , Animais , Automação , Biologia Computacional/métodos , Genômica/métodos , Camundongos , Microinjeções/métodos , Software , Fluxo de TrabalhoRESUMO
In mice, ovarian stimulation via hormone administration is an effective method for obtaining many ova simultaneously, but its effect is reduced by the influence of aging. In this study, we demonstrate that this problem can be improved by administering the gonadotropin-releasing hormone antagonist Cetrorelix prior to ovarian stimulation. Before 12-month-old female mice were injected with 5 IU pregnant mare serum gonadotropin and 5 IU human chorionic gonadotropin, we administered 5 µg/kg Cetrorelix for 7 consecutive days (7 times) or 3 times once every 3 days. As a result, 8.7 ± 1.9 (mean ± SEM, n=10) and 9.8 ± 1.3 (n=10) oocytes were obtained, respectively, as opposed to 4.7 ± 1.2 oocytes (n=9) in the case of no administration. Collagen staining of ovarian tissue showed that Cetrorelix administration reduced the degree of fibrosis, which improved ovarian function. In addition, equivalent fertilization and fetal development rates between control and Cetrorelix-treated aged mouse-derived oocytes were confirmed by in vitro fertilization and embryo transfer (Fertilization rate; control: 92.2% vs. 3 times: 96.9%/7 times: 88.5%, Birth rate; control: 56.4% vs. 3 times: 58.3%/7 times: 51.8%), indicating the normality of the obtained oocytes. It is concluded that Cetrorelix improved the effect of superovulation in aged mice without reducing oocyte quality. This procedure will contribute to animal welfare by extending the effective utilization of aged female breeding mice.
Assuntos
Envelhecimento/fisiologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/administração & dosagem , Camundongos/fisiologia , Superovulação/efeitos dos fármacos , Envelhecimento/patologia , Bem-Estar do Animal , Animais , Cruzamento , Feminino , Fibrose/prevenção & controle , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/farmacologia , Antagonistas de Hormônios/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ovário/patologia , Ovário/fisiologia , Indução da Ovulação/métodosRESUMO
We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Autofagia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Hipocampo/metabolismo , Proteínas de Membrana Transportadoras/genética , Neocórtex/metabolismo , Medula Espinal/metabolismo , Vacúolos/metabolismo , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Corpos Multivesiculares/metabolismo , Neocórtex/patologia , Medula Espinal/patologia , Vacúolos/patologiaRESUMO
Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.
Assuntos
Envelhecimento/genética , Regulação da Expressão Gênica/genética , Hematopoese/genética , Sistema Hematopoético/fisiologia , Código das Histonas/genética , Histona Desmetilases/fisiologia , Animais , Senescência Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Predisposição Genética para Doença , Hematopoese Extramedular , Histona Desmetilases/deficiência , Histona Desmetilases/genética , Reconstituição Imune , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Experimental/genética , Leucemia Experimental/virologia , Masculino , Camundongos , Camundongos Knockout , Vírus da Leucemia Murina de Moloney/fisiologia , Células Mieloides/patologia , Quimera por Radiação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Integração ViralRESUMO
Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/ß-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Células-Tronco/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O3/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Recidiva Local de Neoplasia/metabolismo , Diester Fosfórico Hidrolases/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , beta Catenina/metabolismoRESUMO
BRAFV600E mutation accounts for up to 90% of all BRAF mutations in human colorectal cancer (CRC), and constitutively activates the MEK-MAPK pathway. It is recognized that neutralizing mAbs for epidermal growth factor receptor alone are not effective for CRC with BRAFV600E mutation. Therefore, there is increasing interest in identification of the possible therapeutic targets in downstream of BRAF mutation in CRCs. To address this, we studied genome engineered mouse models for colonic neoplasia that has BrafV600E mutation on the basis of Apc inactivation, induced in 2 distinct Cre mouse models, CDX2P-G22Cre and CDX2P-CreERT2 mice. We carried out oligonucleotide microarray analysis for colonic neoplasia generated in these mouse models, and compared gene expression profiles among Kras/Braf WT, Kras-mutated, and Braf-mutated mouse colon tumors to seek new molecular targets corresponding to the KRAS-BRAF-MAPK axis. We found that the expression of the growth regulation by estrogen in breast cancer protein 1 (Greb1) was the most upregulated gene in Braf-mutated mouse tumors compared to Kras/Braf WT counterparts. The silencing of GREB1 significantly reduced the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GREB1 promoted cell proliferation. Although GREB1 was first identified as a hormone-responsive gene mediating estrogen-stimulated cell proliferation in endometriosis, breast, and ovarian cancers, these results suggest that RAS-RAF-MAPK signaling upregulates GREB1 expression in CRC, resulting in cellular proliferation. Thus, GREB1 is a possible therapeutic target for CRCs with BrafV600E mutation.