RESUMO
Sepsis-associated encephalopathy (SAE) is a critical neurological complication of sepsis and represents a crucial factor contributing to high mortality and adverse prognosis in septic patients. This study explored the contribution of NAT10-mediated messenger RNA (mRNA) acetylation in cognitive dysfunction associated with SAE, utilizing a cecal ligation and puncture (CLP)-induced SAE mouse model. Our findings demonstrate that CLP significantly upregulates NAT10 expression and mRNA acetylation in the excitatory neurons of the hippocampal dentate gyrus (DG). Notably, neuronal-specific Nat10 knockdown improved cognitive function in septic mice, highlighting its critical role in SAE. Proteomic analysis, RNA immunoprecipitation, and real-time qPCR identified GABABR1 as a key downstream target of NAT10. Nat10 deletion reduced GABABR1 expression, and subsequently weakened inhibitory postsynaptic currents in hippocampal DG neurons. Further analysis revealed that microglia activation and the release of inflammatory mediators lead to the increased NAT10 expression in neurons. Microglia depletion with PLX3397 effectively reduced NAT10 and GABABR1 expression in neurons, and ameliorated cognitive dysfunction induced by SAE. In summary, our findings revealed that after CLP, NAT10 in hippocampal DG neurons promotes GABABR1 expression through mRNA acetylation, leading to cognitive dysfunction.
Assuntos
Disfunção Cognitiva , RNA Mensageiro , Encefalopatia Associada a Sepse , Animais , Acetilação , Encefalopatia Associada a Sepse/metabolismo , Encefalopatia Associada a Sepse/genética , Camundongos , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Masculino , Acetiltransferases/metabolismo , Acetiltransferases/genética , Neurônios/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Sepse/metabolismo , Sepse/complicações , Sepse/genética , Microglia/metabolismo , Hipocampo/metabolismo , Giro Denteado/metabolismoRESUMO
The classical manifestation of COVID-19 is pulmonary infection. After host cell entry via human angiotensin-converting enzyme II (hACE2), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can infect pulmonary epithelial cells, especially the AT2 (alveolar type II) cells that are crucial for maintaining normal lung function. However, previous hACE2 transgenic models have failed to specifically and efficiently target the cell types that express hACE2 in humans, especially AT2 cells. In this study, we report an inducible, transgenic hACE2 mouse line and showcase three examples for specifically expressing hACE2 in three different lung epithelial cells, including AT2 cells, club cells, and ciliated cells. Moreover, all these mice models develop severe pneumonia after SARS-CoV-2 infection. This study demonstrates that the hACE2 model can be used to precisely study any cell type of interest with regard to COVID-19-related pathologies.
Assuntos
COVID-19 , Humanos , Animais , Camundongos , Camundongos Transgênicos , SARS-CoV-2 , Células Epiteliais , Células Epiteliais Alveolares , Modelos Animais de DoençasRESUMO
The Gt(ROSA)26Sor ( ROSA26) and H11 loci are commonly used as safe harbors for the construction of targeted transgenic mice. However, it remains unclear whether these two loci have distinct effects on transgene expression. In this study, we insert three differently colored fluorescent protein expression cassettes (EGFP, tdTomato and mTagBFP2) driven by the CAG promoter into the ROSA26 and H11 loci. We generate five single-transgenic mouse models and a triple-transgenic mouse model expressing three distinct fluorescent proteins simultaneously. Our results reveal that the efficiency of transgene expression is greater at the ROSA26 locus with a reverse orientation relative to the transcription of the ROSA26 gene. In most tissues examined, the efficiency of transgene expression at the ROSA26 locus exceeds that at the H11 locus. Moreover, the expression profiles of identical transgenes display discrepancies across various tissues, and notably, substantial heterogeneity in transgene expression is discernible within cells of the same tissue. Our findings offer a valuable reference for the selection of safe harbors and strategies for the construction of transgenic mouse models.
RESUMO
CD8+ T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8+T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8aem(IRES-AkaLuci-2A-EGFP) knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8+ T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8+ T cell biology in experimental applications and preclinical translational studies.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Neoplasias do Colo/diagnóstico por imagem , Efeito Fundador , Edição de Genes/métodos , Genoma , Camundongos Transgênicos/genética , Animais , Antígenos CD8/genética , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Diagnóstico por Imagem/métodos , Técnicas de Introdução de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Xenoenxertos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos/imunologia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Zigoto/imunologia , Zigoto/metabolismoRESUMO
Neuron-restrictive silencer factor (NRSF) is a zinc finger protein that acts as a negative transcriptional regulator by recruiting histone deacetylases and other co-factors. It plays a crucial role in nervous system development and is recently reported to be involved in tumorigenesis in a tumor type-dependent manner; however, the role of NRSF in hepatocellular carcinoma (HCC) tumorigenesis remains unclear. Here, we found that NRSF expression was up-regulated in 27 of 49 human HCC tissue samples examined. Additionally, mice with conditional NRSF-knockout in the liver exhibited a higher tolerance against diethylnitrosamine (DEN)-induced acute liver injury and were less sensitive to DEN-induced HCC initiation. Our results showed that silencing NRSF in HepG2 cells using RNAi technology significantly inhibited HepG2 cell proliferation and severely hindered their migration and invasion potentials. Our results demonstrated that NRSF plays a pivotal role in promoting DEN-induced HCC initiation via a mechanism related to the STAT3 and AKT signaling pathways. Thus, NRSF could be a potential therapeutic target for treating human HCC.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Carcinogênese , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proliferação de Células/genética , Dietilnitrosamina/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para CimaRESUMO
The relationship of carcinogenesis and DNA methyltransferases has attracted extensive attention in tumor research. We reported previously that inhibition of de novo DNA methyltransferase 3a (Dnmt3a) in murine B16 melanoma cells significantly suppressed tumor growth and metastasis in xenografted mouse model. Here, we further demonstrated that knockdown of Dnmt3a enhanced the proliferation in anchor-independent conditions of B16 cells, but severely disrupted its multipotent differentiation capacity in vitro. Furthermore, transforming growth factor ß1, a key trigger in stem cell differentiation and tumor cell epithelial-mesenchymal transition (EMT), mainly induced apoptosis, but not EMT in Dnmt3a-deficient B16 cells. These data suggested that Dnmt3a is required for maintaining the tumor stemness of B16 cells and it assists B16 cells to escape from death during cell differentiation. Thus it is hypothesized that not only extraordinary self-renewal ability, but also the capacity of multipotent differentiation is necessary for the melanoma tumorigenesis. Inhibition of multipotent differentiation of tumor cells may shed light on the tumor treatment.
Assuntos
Carcinogênese/metabolismo , DNA (Citosina-5-)-Metiltransferases/fisiologia , Melanoma Experimental/patologia , Células-Tronco Neoplásicas/patologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , DNA Metiltransferase 3A , Transição Epitelial-Mesenquimal , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Utilizing cytotoxic proteins linked to tumor targeting molecules as anti-tumor drugs is a promising approach. However, most cytotoxins derived from bacteria or plants have inherent problems such as large molecular weights and they trigger a strong immune system reaction, which leads to drug failure and serious side effects. Amphinase (Amph) is a ribonuclease with a low molecular weight that is found in northern leopard frog oocytes. It has strong cytotoxicity against tumor cell lines in vitro and weak immunogenicity in vivo, and is a promising candidate in the development of targeted drugs. Transforming growth factor-α (TGF-α) that binds to the epidermal growth factor receptor (EGFR) is being used as a targeting molecule for the treatment of EGFR high-expressing tumors. In this study, we expressed and purified a recombinant amphinase and its TGF-α fusion protein (AGT) separately from Escherichia coli. AGT exhibited more significant cytotoxicity in vitro on EGFR high-expressing tumor cell lines, and stronger anti-tumor effects in vivo. This fusion protein also exhibited unusual thermostability, low in vivo immunogenicity, and side effects. Our results provide a new entry point for the development of novel, highly efficient anti-tumor targeting biological agents with low immunogenicity.
Assuntos
Antineoplásicos/química , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Ribonucleases/química , Células A549 , Animais , Apoptose , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Feminino , Células HL-60 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Oócitos/metabolismo , Plasmídeos/metabolismo , Ranidae , Proteínas Recombinantes de Fusão/química , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Onconase (Onc) is a cytotoxic ribonuclease derived from leopard frog oocytes or early embryos, and has been applied to the treatment of malignant mesothelioma in clinics. Onc also exhibits effective growth suppression of human non-small-cell lung cancer (NSCLC). Artemisinin (Art) and its derivatives are novel antimalarial drugs that exhibit antitumor and antivirus activities. In this study, we investigated the antitumor effects of combinations of Onc and an Art derivative, dihydroartemisinin (DHA), both in vitro and in vivo Isobologram analyses showed synergistic effects on the proliferation of NSCLC cells under the treatment with Onc and DHA. In vivo experiments also showed that the antitumor effect of Onc was markedly enhanced by DHA in mouse xenograft models. No obvious adverse effect was observed after the treatment. The density of microvasculature in the tumor tissues treated with Onc/DHA combination was lower than those treated with Onc or DHA alone. The above results are consistent with the results of the matrigel plug test for angiogenesis suppression using the Onc/DHA combination. These results imply that the anti-angiogenesis effects may make important contributions to the in vivo antitumor effects of the Onc/DHA combination treatment. The Onc/DHA combination therapy may have the potential to become a novel regimen for NSCLC and mesothelioma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Células A549 , Animais , Antimaláricos/administração & dosagem , Antimaláricos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Artemisininas/administração & dosagem , Artemisininas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/irrigação sanguínea , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/metabolismo , Ribonucleases/administração & dosagem , Ribonucleases/farmacologia , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is the most common abdominal malignant tumor in childhood. Immunotoxin (IT) that targets the tumor cell surface receptor is a new supplementary therapeutic treatment approach. The purpose of this study is to detect the expression of epidermal growth factor receptor (EGFR) in neuroblastoma cell lines and tissues, and to explore if IT therapy can be used to treat refractory neuroblastoma. The EGFR expression in human neuroblastoma tissue samples was detected by immunohistochemistry staining. The positive rate of EGFR expression was 81.0% in neuroblastoma tissue and 50.0% in gangliocytoma, respectively, but without statistical significance between them (P > 0.05). The positive rate of EGFR expression in favorable type and unfavorable type was 62.5% and 92.3%, respectively, but they were not statistically different (P > 0.05). Results from pre-chemotherapy and post-chemotherapy samples showed that there was no significant statistical difference (P > 0.05) between them in the EGFR expression. Furthermore, the EGFR expression levels in five neuroblastoma cell lines were measured using cell-based ELISA assay and western blot analysis. The results showed that the expression of EGFR was higher in KP-N-NS and BE(2)-C than those in other cell lines. Our results revealed that there are consistent and widespread expressions of EGFR in neuroblastoma tissues as well as in neuroblastoma cell lines, suggesting that it is possible to develop future treatment strategies of neuroblastoma by targeting at the EGFR.
Assuntos
Receptores ErbB/metabolismo , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Neuroblastoma/patologiaRESUMO
Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene's promoter region turns the gene on and off in a tetracycline-inducible manner. Nmyc(TRE) mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. Nmyc(TRE) mice were crossed to tTS transgenic mice to produce Nmyc(TRE/TRE): tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS-dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.
Assuntos
Regulação da Expressão Gênica , Marcação de Genes/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Córtex Cerebelar/anormalidades , Córtex Cerebelar/crescimento & desenvolvimento , Córtex Cerebelar/metabolismo , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Genes Letais , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta , Sindactilia/etiologiaRESUMO
With the postponement of female reproductive age and the higher incidence of cancer in young people, fertility preservation has become increasingly important in childbearing age. Chemotherapy during pregnancy is crucial for maternal cancer treatments and fetal outcomes. It is a need to further study ovarian damage caused by chemotherapy drug combinations and long-term effects on offspring development, and a detailed understanding of side effects of chemotherapy drugs. In this study, chemotherapy drug combinations significantly impacted on ovarian function, especially epirubicin/cyclophosphamide (EC) combination led to an unbalance in the development of the left and right ovary. Exposure to EC and cisplatin/paclitaxel (TP) increased the number of progenitor follicles while decreased the count of antral follicles and corpora luteum. As to the estrus cycle, EC exposure resulted in a longer estrus period and diestrus period, while TP exposure only extended the diestrus period. EC and TP affected steroid biosynthesis by reducing the expression of SF1 and P450arom.γ-H2AX was detected in both EC and TP exposure groups. As to the impact on the offspring from 4T1 tumor-bearing pregnant mice injected with EC, no significant difference was observed in the physical and neurological development compared to the control, but the ovarian weights, estrus cycles of the offspring were significantly different. Chemotherapy drug combinations exhibit ovarian toxicity, not only causing direct damage on the follicle cells but also disrupting steroid biosynthesis. The reproductive system of offspring from maternal tumor-bearing mice exposed to chemotherapy drugs was observed disorder, but the concrete mechanism still needs further exploration.
Assuntos
Cisplatino , Ciclofosfamida , Epirubicina , Ovário , Feminino , Animais , Ciclofosfamida/toxicidade , Ciclofosfamida/efeitos adversos , Gravidez , Ovário/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Epirubicina/efeitos adversos , Epirubicina/toxicidade , Paclitaxel/efeitos adversos , Paclitaxel/toxicidade , Camundongos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Camundongos Endogâmicos BALB C , Antineoplásicos/efeitos adversos , Antineoplásicos/toxicidadeRESUMO
BACKGROUND: Pulmonary fibrosis (PF), the most common clinical type of irreversible interstitial lung disease with one of the worse prognoses, has a largely unknown molecular mechanisms that underlies its progression. CD5 molecule-like (CD5L) functions in an indispensable role during inflammatory responses; however, whether CD5L functions in regulating bleomycin (BLM)-induced lung fibrosis is less clear. METHODS: Herein, we describe the engineering of Cd5l knockout mice using CRISPR/Cas9 gene editing technology. The BLM-induced model of acute lung injury represents the most widely used experimental rodent model for PF. RESULTS: Taking advantage of this model, we demonstrated that both CD5L mRNA and protein were enriched in the lungs of mice following BLM-induced pulmonary fibrosis. Inhibition of CD5L prevented mice from BLM-induced lung fibrosis and injury. In particular, a lack of CD5L significantly attenuated inflammatory response and promoted M2 polarization in the lung of this pulmonary fibrosis model as well as suppressing macrophage apoptosis. CONCLUSIONS: Collectively, our data support that CD5L deficiency can suppress the development of pulmonary fibrosis, and also provides new molecular targets for the use of immunotherapy to treat lung fibrosis.
Assuntos
Fibrose Pulmonar , Animais , Camundongos , Bleomicina/efeitos adversos , Citocinas/metabolismo , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/prevenção & controleRESUMO
PURPOSE: To induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study. METHODS: A loxP-cassette vector was constructed comprising a sequence of loxP -Puro-3∗polyA-loxP, followed by amphinase cDNA. The vector was transfected into neuroblastoma cell lines, SK-N-BE(2)-C, by Lipofectamine LTX. The transfected cells were selected by puromycin for two weeks. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) were conducted to verify that the loxP-cassette vector was stably transfected. The expression of amphinase was activated by the addition of Cre recombinase delivered by a lentiviral vector and identified by qPCR and Western blotting (WB). CCK8 assay and colony formation assay were conducted to check the effect of amphinase on cell proliferation. RNA sequencing (RNA-seq) was conducted to explore the targeted pathway of Cre/loxP-mediated amphinase and recombinant amphinase. RESULTS: Stably transfected cell clones were achieved through puromycin selection. After Cre recombinase was delivered to the cells, the loxP-flanked fragment was deleted and the expression of amphinase was induced, which were tested by PCR and qPCR. It was shown that cell proliferation was significantly inhibited by the amphinase mediated by the Cre/loxP system. KEGG enrichment and GSEA analysis indicated that amphinase had an impact on the ER function of neuroblastoma cells, which was identical to the effect of the recombinant amphinase. CONCLUSION: We successfully induce the expression of amphinase in neuroblastoma cell lines via Cre/loxP system. The Cre/loxP-mediated amphinase had a similar antitumor mechanism to the recombinant amphinase, providing a powerful tool for the mechanism study of amphinase.
Assuntos
Vetores Genéticos , Neuroblastoma , Humanos , Integrases/genética , Integrases/metabolismo , Linhagem Celular , Neuroblastoma/genéticaRESUMO
A ratiometric and pH-sensitive fluorescent dye named IDE was applied to the detection of argine and lysine from common amino acids and exploited to monitor the Lys and Arg levels in living cells and zebrafish larvae successfully. IDE will be a useful fluorescence indicator of pH changes by Lys and Arg.
Assuntos
Corantes Fluorescentes , Peixe-Zebra , Humanos , Animais , Corantes Fluorescentes/química , Peixe-Zebra/metabolismo , Células HeLa , Lisina/metabolismo , Espectrometria de Fluorescência/métodosRESUMO
The peroxiredoxin (PRDX) family is a class of antioxidant enzymes with peroxidase activity. Human PRDXs currently have six members (PRDX1-6), which are gradually becoming potential therapeutic targets for major diseases such as cancer. In this study, we reported ainsliadimer A (AIN), a sesquiterpene lactone dimer with antitumor activity. We found that AIN directly targets Cys173 of PRDX1 and Cys172 of PRDX2 and then inhibits their peroxidase activities. As a result, the level of intracellular ROS increases, causing oxidative stress damage in mitochondria, inhibiting mitochondrial respiration, and significantly inhibiting ATP production. AIN inhibits the proliferation and induces apoptosis of colorectal cancer cells. Additionally, it inhibits tumor growth in mice and the growth of tumor organoid models. Therefore, AIN can be one of the natural compounds targeting PRDX1 and PRDX2 in the treatment of colorectal cancer.
Assuntos
Neoplasias Colorretais , Peroxirredoxinas , Animais , Humanos , Camundongos , Antioxidantes , Apoptose , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Espécies Reativas de OxigênioRESUMO
Unraveling cell fate plasticity during tissue homeostasis and repair can reveal actionable insights for stem cell biology and regenerative medicine. In the pancreas, it remains controversial whether lineage transdifferentiation among the exocrine cells occur under pathophysiological conditions. Here, to address this question, we used a dual recombinase-mediated genetic system that enables simultaneous tracing of pancreatic acinar and ductal cells using two distinct genetic reporters, avoiding the "ectopic" labeling by Cre-loxP recombination system. We found that acinar-to-ductal transdifferentiation occurs after pancreatic duct ligation or during caerulein-induced pancreatitis, but not during homeostasis or after partial pancreatectomy. On the other hand, pancreatic ductal cells contribute to new acinar cells after significant acinar cell loss. By genetic tracing of cell proliferation, we also quantify the cell proliferation dynamics and deduce the turnover rate of pancreatic exocrine lineages during homeostasis. Together, these results suggest that the lineage transdifferentiation happens between acinar cells and ductal cells in the pancreatic exocrine glands under specific conditions.
RESUMO
A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.
RESUMO
After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.
Assuntos
Cardiopatias , Fibroblastos/metabolismo , Cardiopatias/genética , Cardiopatias/patologia , Fibrose/genética , Animais , Camundongos , Envelhecimento , Proliferação de Células , Via de Sinalização Wnt , Camundongos TransgênicosRESUMO
Emerging evidence suggests that pyroptosis is involved in sepsis. However, the role of neutrophil pyroptosis in sepsis and the mechanisms remains elusive. We find that N-acetyltransferase 10 (NAT10), an acetyltransferase responsible for the N4-acetylation of Cytidine (ac4C) in mRNA, is significantly downregulated in neutrophils from septic mice. Neutrophil-specific over-expression of NAT10 improves the survival and ameliorates lung injury in septic mice by inhibiting neutrophil pyroptosis. Notably, UNC-52-like kinase 1 (ULK1) is identified as the target of NAT10 in neutrophils. The decreased expression of NAT10 resultes in the decay of ULK1 transcripts and therefore the reduced expression of ULK1. As a regulator of STING phosphorylation, the loss of ULK1 enhances the activation of STING-IRF3 signaling and subsequently the elevated pyroptosis-inducing NLRP3 inflammasome in neutrophils. While over-expression of NAT10 restrains pyroptosis in neutrophils as well as septic lethality in mice by reversing the ULK1-STING-NLRP3 axis. The decreased expression of NAT10 are also observed in sepsis patients and its correlation with clinical severity is found. Collectively, our findings disclose that NAT10 is a negative regulator of neutrophil pyroptosis and its downregulation contributes to the progress of sepsis by exacerbating pyroptosis via the ULK1-STING-NLRP3 axis, therefore revealing a potential therapeutic target for sepsis.