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1.
Cell Death Dis ; 11(1): 39, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959745

RESUMO

Ginsenosides exhibit a large variety of biological activities in maintaining physical health; however, the molecule underpinnings underlining these biological activities remain to be defined. Here, we took a cellular condition that compound K (CK) induces autophagic cell death in HeLa cells, and setup a high-throughput genetic screening using CRISPR technology. We have identified a number of CK-resistant and CK-sensitive genes, and further validated PMAIP1 as a CK-resistant gene and WASH1 as a CK-sensitive gene. Compound K treatment reduces the expression of WASH1, which further accelerates the autophagic cell death, highlighting WASH1 as an interesting downstream mediator of CK effects. Overall, our study offers an easy-to-adopt platform to study the functional mediators of ginsenosides, and provides a candidate list of genes that are potential targets of CK.

2.
Circulation ; 141(1): 67-79, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31779484

RESUMO

BACKGROUND: Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. METHODS: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, LdlrE208X, based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. CONCLUSIONS: Our work shows that in vivo AAV-CRISPR/Cas9-mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.

3.
Cell Rep ; 29(3): 781-784, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618644

RESUMO

This Matters Arising Response paper addresses the Hoch et al. (2019) Matters Arising paper published concurrently in this issue of Cell Reports. The genetic study in humans revealed a strong association of DNA variants in the SLC16A11 coding region with type 2 diabetes mellitus (T2DM). However, how these T2D variants affect the function of SLC16A11 remains controversial. In Zhao et al. (2019), with studies using genetic knockout mouse models and in vivo gene reconstitution experiments, we demonstrated gain of aberrant functions of mutant SLC16A11-carrying T2D variants, which cause liver steatosis and insulin resistance. Hoch et al. (2019) raise concerns regarding the animal models and experimental settings used in the study. Here, we address their concerns and emphasize that discoveries from the physiological studies of SLC16A11 by using mouse models disagree with the previous proposal by Rusu et al. (2017) that "therapeutics that enhance SLC16A11 levels or activity may be beneficial for T2D."

4.
Nat Commun ; 10(1): 4158, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519886

RESUMO

The ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing RIPK1K376R which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1K376R are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1K376R/K376R lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1-/- Ripk1K376R/K376R mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity-dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally.


Assuntos
Sobrevivência Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Desenvolvimento Embrionário/genética , Feminino , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Inflamação/genética , Inflamação/metabolismo , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação/genética
5.
World J Stem Cells ; 11(8): 535-547, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31523372

RESUMO

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years' efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro. This has offered a new paradigm for genome-wide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.

6.
Oral Dis ; 25(7): 1789-1797, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31283861

RESUMO

BACKGROUND: Porphyromonas gingivalis is the main pathogen of periodontal disease affecting over half of the worldwide adult population. Recent studies have shown that P. gingivalis is related to the development of non-alcoholic fatty liver disease (NAFLD), a global major chronic liver disease, especially in developed countries. However, how P. gingivalis contributes to the pathogenesis of NAFLD has not been fully clarified. We aimed to conduct a preliminary exploration of the underlying mechanism of P. gingivalis infection in the development of NAFLD. METHODS: Human hepatocellular cells HepG2 were incubated with/without oleic acid (OA) and tested for lipid accumulation upon stimulation by lipopolysaccharide (LPS) derived from P. gingivalis or Escherichia coli. Intracellular lipid droplet formation was analyzed and quantified by Oil Red O staining. The involvement of signaling pathway molecules and pro-inflammatory cytokines related to NF-κB and MAPKs were examined with Western blot and quantitative real-time PCR (qRT-PCR) analyses and further evaluated with inhibitor treatment and RNA interference. RESULTS: HepG2 cells accumulated more intracellular lipids when stimulated with P. gingivalis LPS, as compared to cells treated with E. coli LPS or control. Further pathway analysis demonstrated that after stimulation with P. gingivalis LPS, cells displayed significantly upregulated MyD88 expression, increased phosphorylation of p65 and JNK, and more release of pro-inflammatory cytokines, such as IL-1, IL-8, and TNF-α. In addition, suppression of phosphorylation of p65 and JNK by inhibitors and RNA interference resulted in a reduction in lipid accumulation upon P. gingivalis LPS treatment. CONCLUSIONS: These results suggest that P. gingivalis-derived LPS may contribute to intracellular lipid accumulation and inflammatory reaction of HepG2 cells via the activation of NF-κB and JNK signaling pathways. This study offers a possible explanation to the functional involvement of P. gingivalis infection in the pathological progression of NAFLD. These findings may help design new treatment strategies in NAFLD.


Assuntos
Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases , NF-kappa B , Hepatopatia Gordurosa não Alcoólica/patologia , Periodontite/microbiologia , Porphyromonas gingivalis , Adulto , Infecções por Bacteroidaceae , Western Blotting , Humanos , Hepatopatia Gordurosa não Alcoólica/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real
7.
Theranostics ; 9(12): 3501-3514, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281493

RESUMO

Identification of proper agents to increase or activate UCP1+ cells in adipose tissues remains a potent therapeutic strategy to combat obesity. Screening systems for UCP1 activators have been previously established and allow for unbiased discovery of effective compound(s). Methods: A previously established Ucp1-2A-GFP reporter system was applied to a chemical library containing 33 phosphatase inhibitors. Compounds that can significantly activate UCP1 expression were further tested in vivo in mouse adipose tissues. Possible underlying mechanism was explored via RNA profiling, CMAP analysis, CRISPR targeting as well as inhibitor treatments. Results: We identified BML-260, a known potent inhibitor of the dual-specific phosphatase JSP-1, that significantly increased UCP1 expression in both brown and white adipocytes. BML-260 treatment also activated oxidative phosphorylation genes, increased mitochondrial activity as well as heat generation in vitro and in vivo. Mechanistic studies revealed that effect of BML-260 on adipocytes was partly through activated CREB, STAT3 and PPAR signaling pathways, and was unexpectedly JSP-1 independent. Conclusion: The rhodanine derivate BML-260 was previously identified to be a JSP-1 inhibitor, and thus was proposed to treat inflammatory and proliferative disorders associated with dysfunctional JNK signaling. This work provides evidences that BML-260 can also exert a JSP-1-independent effect in activating UCP1 and thermogenesis in adipocytes, and be potentially applied to treat obesity.

9.
Cell Rep ; 26(4): 884-892.e4, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673611

RESUMO

DNA variants in the SLC16A11 coding region were identified to be strongly associated with type 2 diabetes (T2DM) in a Mexican population. Previous studies suggested that these variants disrupt SLC16A11 function and therefore proposed to revive SLC16A11 levels or activity to achieve therapeutic benefit. However, with knockout mouse models, here we show that Slc16a11 depletion has no significant metabolic defects. Further studies demonstrate that reconstitution of the mutant, but not the wild-type Slc16a11, in the liver of knockout mice causes more triglyceride accumulation and induction of insulin resistance via upregulation of lipin 1, suggesting gaining of aberrant functions of the mutant protein that affects lipid metabolism. Our findings offer a different explanation to the function of these diabetic variants, challenging the concept of enhancing SLC16A11 function to treat T2DM. The contradictory results by our and previous studies suggest that how the SLC16A11 locus contributes to human metabolism warrants further investigation.

10.
J Clin Invest ; 129(3): 972-987, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30688657

RESUMO

Metastasis is the dominant cause of patient death in small-cell lung cancer (SCLC), and a better understanding of the molecular mechanisms underlying SCLC metastasis may potentially improve clinical treatment. Through genome-scale screening for key regulators of mouse Rb1-/- Trp53-/- SCLC metastasis using the pooled CRISPR/Cas9 library, we identified Cullin5 (CUL5) and suppressor of cytokine signaling 3 (SOCS3), two components of the Cullin-RING E3 ubiquitin ligase complex, as top candidates. Mechanistically, the deficiency of CUL5 or SOCS3 disrupted the functional formation of the E3 ligase complex and prevented the degradation of integrin ß1, which stabilized integrin ß1 and activated downstream focal adhesion kinase/SRC (FAK/SRC) signaling and eventually drove SCLC metastasis. Low expression levels of CUL5 and SOCS3 were significantly associated with high integrin ß1 levels and poor prognosis in a large cohort of 128 clinical patients with SCLC. Moreover, the CUL5-deficient SCLCs were vulnerable to the treatment of the FDA-approved SRC inhibitor dasatinib. Collectively, this work identifies the essential role of CUL5- and SOCS3-mediated integrin ß1 turnover in controlling SCLC metastasis, which might have therapeutic implications.

11.
Mol Cell ; 72(2): 380-394.e7, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30293782

RESUMO

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citidina Desaminase/genética , Processamento de RNA/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Distrofina/genética , Éxons/genética , Redes Reguladoras de Genes , Células HEK293 , Humanos , Íntrons/genética , Camundongos , Fases de Leitura Aberta/genética , Sítios de Splice de RNA/genética
12.
EBioMedicine ; 37: 344-355, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30348622

RESUMO

BACKGROUND: The pharmacological activation of thermogenesis in brown adipose tissue has long been considered promising strategies to treat obesity. However, identification of safe and effective agents remains a challenge. In this study, we addressed this challenge by developing a cellular system with a fluorescence readout, and applied in a high-throughput manner to screen for FDA-approved drugs that may activate endogenous UCP1 expression in adipocytes. METHODS: We have generated a Ucp1-2A-GFP reporter mouse, in which GFP intensity serves as a surrogate of the endogenous expression level of UCP1 protein; and immortalized brown adipocytes were derived from this mouse model and applied in drug screening. Candidate drugs were further tested in mouse models either fed with normal chow or high fat diet to induce obesity. FINDINGS: By using the cellular screening platform, we identified a group of FDA-approved drugs that can upregulate UCP1 expression in brown adipocyte, including previously known UCP1 activators and new candidate drugs. Further studies focusing on a previously unreported drug-sutent, revealed that sutent treatment could increase the energy expenditure and inhibit lipid synthesis in mouse adipose and liver tissues, resulting in improved metabolism and resistance to obesity. INTERPRETATION: This study offered an easy-to-use cellular screening system for UCP1 activators, and provided a candidate list of FDA-approved drugs that can potentially treat obesity. Further study of these candidates may shed new light on the drug discovery towards obesity. FUND: National Key Research and Development Program and the Strategic Priority Research Program of the Chinese Academy of Sciences, etc. (250 words).


Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Desacopladora 1/biossíntese , Adipócitos Marrons/patologia , Tecido Adiposo Marrom/patologia , Animais , Linhagem Celular Transformada , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Camundongos , Camundongos Transgênicos , Proteína Desacopladora 1/genética , Estados Unidos , United States Food and Drug Administration
13.
Proc Natl Acad Sci U S A ; 115(46): E10849-E10858, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30373812

RESUMO

The metabolic regulation of cell death is sophisticated. A growing body of evidence suggests the existence of multiple metabolic checkpoints that dictate cell fate in response to metabolic fluctuations. However, whether microRNAs (miRNAs) are able to respond to metabolic stress, reset the threshold of cell death, and attempt to reestablish homeostasis is largely unknown. Here, we show that miR-378/378* KO mice cannot maintain normal muscle weight and have poor running performance, which is accompanied by impaired autophagy, accumulation of abnormal mitochondria, and excessive apoptosis in skeletal muscle, whereas miR-378 overexpression is able to enhance autophagy and repress apoptosis in skeletal muscle of mice. Our in vitro data show that metabolic stress-responsive miR-378 promotes autophagy and inhibits apoptosis in a cell-autonomous manner. Mechanistically, miR-378 promotes autophagy initiation through the mammalian target of rapamycin (mTOR)/unc-51-like autophagy activating kinase 1 (ULK1) pathway and sustains autophagy via Forkhead box class O (FoxO)-mediated transcriptional reinforcement by targeting phosphoinositide-dependent protein kinase 1 (PDK1). Meanwhile, miR-378 suppresses intrinsic apoptosis initiation directly through targeting an initiator caspase-Caspase 9. Thus, we propose that miR-378 is a critical component of metabolic checkpoints, which integrates metabolic information into an adaptive response to reduce the propensity of myocytes to undergo apoptosis by enhancing the autophagic process and blocking apoptotic initiation. Lastly, our data suggest that inflammation-induced down-regulation of miR-378 might contribute to the pathogenesis of muscle dystrophy.


Assuntos
MicroRNAs/fisiologia , Músculo Esquelético/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Caspase 9/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Células Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Corrida , Transdução de Sinais , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo
14.
Nat Commun ; 9(1): 3941, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30258187

RESUMO

Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies.


Assuntos
Canalopatias/genética , Canais de Potássio Éter-A-Go-Go/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Canalopatias/tratamento farmacológico , Canalopatias/metabolismo , Modelos Animais de Doenças , Diterpenos/farmacologia , Diterpenos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ésteres de Forbol/farmacologia , Ésteres de Forbol/uso terapêutico , Proteína Quinase C/metabolismo , Triterpenos/farmacologia , Triterpenos/uso terapêutico
16.
Stem Cell Reports ; 11(1): 22-31, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29861165

RESUMO

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.


Assuntos
Diferenciação Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Histona Desacetilases/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Reporter , Genes abl , Fator 4 Nuclear de Hepatócito/metabolismo , Histona Desacetilases/genética , Humanos , Modelos Biológicos , Fenilenodiaminas/farmacologia
17.
Nat Genet ; 50(4): 572-580, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29632379

RESUMO

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.


Assuntos
Adipócitos/patologia , Composição Corporal/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Fatores de Transcrição Sp/genética , Alelos , Animais , Distribuição da Gordura Corporal , Tamanho Celular , Elementos Facilitadores Genéticos , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Impressão Genômica , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Lipogênese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fatores de Risco , Caracteres Sexuais
18.
J Biol Chem ; 293(18): 6883-6892, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29507093

RESUMO

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Desoxirribonuclease I/metabolismo , Edição de Genes , Terapia Genética/métodos , Tirosinemias/genética , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Células HEK293 , Hepatócitos/citologia , Humanos , Hidrolases/genética , Mutação INDEL , Cirrose Hepática/etiologia , Cirrose Hepática/prevenção & controle , Masculino , Ratos , Tirosinemias/complicações , Tirosinemias/imunologia , Tirosinemias/terapia
19.
Nanotoxicology ; 11(6): 737-750, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28669258

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO2 NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO2 NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO2 NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO2 NPs (10 and 50 nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO2 NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO2 NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO2 NPs. These results demonstrate that TiO2 NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.


Assuntos
Imunidade Inata/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanopartículas/toxicidade , Titânio/toxicidade , Animais , Linhagem Celular , Citocinas/sangue , Citocinas/imunologia , Imunidade Inata/genética , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Nanopartículas/química , Choque Séptico/sangue , Choque Séptico/imunologia , Titânio/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
20.
Cell Death Differ ; 24(8): 1459-1469, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28574501

RESUMO

RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd-/- embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1K45A/K45A mouse line as previously reported and a novel Rip1Δ/Δ mouse line with an altered P-loop in the kinase domain. While RIP1K45A could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1Δ rescued lethality of Fadd-/- mice at E11.5 and Fadd-/-Rip1Δ/Δ mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1K45A/K45A cells, Rip1Δ/Δcells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.


Assuntos
Desenvolvimento Embrionário/genética , Proteína de Domínio de Morte Associada a Fas/genética , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Embrião de Mamíferos , Proteína de Domínio de Morte Associada a Fas/deficiência , Feminino , Proteínas Ativadoras de GTPase/deficiência , Genes Letais , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Gravidez , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais
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