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1.
Hypertension ; 76(3): 827-838, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32683902

RESUMO

NOX5 (NADPH oxidase 5) is a homolog of the gp91phox subunit of the phagocyte NOX, which generates reactive oxygen species. NOX5 is involved in sperm motility and vascular contraction and has been implicated in diabetic nephropathy, atherosclerosis, and stroke. The function of NOX5 in the cardiac hypertrophy is unknown. Because NOX5 is a Ca2+-sensitive, procontractile NOX isoform, we questioned whether it plays a role in cardiac hypertrophy. Studies were performed in (1) cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure, (2) NOX5-expressing rat cardiomyocytes, and (3) mice expressing human NOX5 in a cardiomyocyte-specific manner. Cardiac hypertrophy was induced in mice by transverse aorta coarctation and Ang II (angiotensin II) infusion. NOX5 expression was increased in human failing hearts. Rat cardiomyocytes infected with adenoviral vector encoding human NOX5 cDNA exhibited elevated reactive oxygen species levels with significant enlargement and associated increased expression of ANP (atrial natriuretic peptides) and ß-MHC (ß-myosin heavy chain) and prohypertrophic genes (Nppa, Nppb, and Myh7) under Ang II stimulation. These effects were reduced by N-acetylcysteine and diltiazem. Pressure overload and Ang II infusion induced left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction, responses that were exaggerated in cardiac-specific NOX5 trangenic mice. These phenomena were associated with increased reactive oxygen species levels and activation of redox-sensitive MAPK (mitogen-activated protein kinase). N-acetylcysteine treatment reduced cardiac oxidative stress and attenuated cardiac hypertrophy in NOX5 trangenic. Our study defines Ca2+-regulated NOX5 as an important NOX isoform involved in oxidative stress- and MAPK-mediated cardiac hypertrophy and contractile dysfunction.


Assuntos
Acetilcisteína/farmacologia , Cardiomegalia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NADPH Oxidase 5/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Angiotensina II/farmacologia , Animais , Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Sequestradores de Radicais Livres/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fagócitos/enzimologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Vasoconstritores/farmacologia , Miosinas Ventriculares/metabolismo
2.
Hepatology ; 70(5): 1750-1769, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31077413

RESUMO

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/irrigação sanguínea , Proteômica , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
3.
Hepatology ; 70(6): 1942-1957, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30703849

RESUMO

Inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation has emerged as a promising target for the treatment of nonalcoholic steatohepatitis (NASH). Multiple forms of posttranslational modifications determine the activity of ASK1. In addition to phosphorylation, recent studies revealed that ubiquitination is essential for ASK1 activation. However, the endogenous factor that regulates ASK1 ubiquitination and activation remains poorly defined. In this study, we identified the E3 ligase Skp1-Cul1-F-box (SCF) protein F-box/WD repeat-containing protein 5 (FBXW5) as a key endogenous activator of ASK1 ubiquitination. FBXW5 is the central component of the SCF complex (SCFFbxw5 ) that directly interacts with and ubiquitinates ASK1 in hepatocytes during NASH development. An in vivo study showed that hepatocyte-specific overexpression of FBXW5 exacerbated diet-induced systemic and hepatic metabolic disorders, as well as the activation of ASK1-related mitogen-activated protein kinase (MAPK) signaling in the liver. Conversely, hepatocyte-specific deletion of FBXW5 significantly prevented the progression of these abnormalities. Mechanically, FBXW5 facilitated the addition of Lys63-linked ubiquitin to ASK1 and thus exacerbated ASK1-c-Jun N-terminal kinase/p38 MAPK signaling, inflammation, and lipid accumulation. Furthermore, we demonstrated that the N-terminus (S1) and C-terminus (S3) of FBXW5 respectively and competitively ablate the function of FBXW5 on ASK1 activation and served as effective inhibitors of NASH progression. Conclusion: This evidence strongly suggests that SCFFbxw5 is an important activator of ASK1 ubiquitination in the context of NASH. The development of FBXW5(S1) or FBXW5(S3)-mimicking drugs and screening of small-molecular inhibitors specifically abrogating ASK1 ubiquitination-dependent activation are viable approaches for NASH treatment.


Assuntos
Proteínas F-Box/fisiologia , MAP Quinase Quinase Quinase 5/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Ubiquitinação , Animais , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Repetições WD40 , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
5.
Hepatology ; 69(2): 524-544, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29381809

RESUMO

Tumor progression locus 2 (TPL2), a serine/threonine kinase, has been regarded as a potentially interesting target for the treatment of various diseases with an inflammatory component. However, the function of TPL2 in regulating hepatocyte metabolism and liver inflammation during the progression of nonalcoholic fatty liver disease (NAFLD) is poorly understood. Here, we report that TPL2 protein expression was significantly increased in fatty liver from diverse species, including humans, monkeys, and mice. Further investigations revealed that compared to wild-type (WT) littermates, hepatocyte-specific TPL2 knockout (HKO) mice exhibited improved lipid and glucose imbalance, reserved insulin sensitivity, and alleviated inflammation in response to high-fat diet (HFD) feeding. Overexpression of TPL2 in hepatocytes led to the opposite phenotype. Regarding the mechanism, we found that mitogen-activated protein kinase kinase 7 (MKK7) was the specific substrate of TPL2 for c-Jun N-terminal kinase (JNK) activation. TPL2-MKK7-JNK signaling in hepatocytes represents a promising drugable target for treating NAFLD and associated metabolic disorders. Conclusion: In hepatocytes, TPL2 acts as a key mediator that promotes both liver and systemic metabolic disturbances by specifically increasing MKK7-JNK activation.


Assuntos
Hepatócitos/metabolismo , Inflamação/metabolismo , Resistência à Insulina , MAP Quinase Quinase Quinases/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Haplorrinos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 7/metabolismo , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Proteínas Proto-Oncogênicas/genética
6.
J Hepatol ; 69(5): 1110-1122, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29958938

RESUMO

BACKGROUND & AIMS: The hepatic injury caused by ischemia/reperfusion (I/R) insult is predominantly determined by the complex interplay of sterile inflammation and liver cell death. Caspase recruitment domain family member 6 (CARD6) was initially shown to play important roles in NF-κB activation. In our preliminary studies, CARD6 downregulation was closely related to hepatic I/R injury in liver transplantation patients and mouse models. Thus, we hypothesized that CARD6 protects against hepatic I/R injury and investigated the underlying molecular mechanisms. METHODS: A partial hepatic I/R operation was performed in hepatocyte-specific Card6 knockout mice (HKO), Card6 transgenic mice with CARD6 overexpression specifically in hepatocytes (HTG), and the corresponding control mice. Hepatic histology, serum aminotransferases, inflammatory cytokines/chemokines, cell death, and inflammatory signaling were examined to assess liver damage. The molecular mechanisms of CARD6 function were explored in vivo and in vitro. RESULTS: Liver injury was alleviated in Card6-HTG mice compared with control mice as shown by decreased cell death, lower serum aminotransferase levels, and reduced inflammation and infiltration, whereas Card6-HKO mice had the opposite phenotype. Mechanistically, phosphorylation of ASK1 and its downstream effectors JNK and p38 were increased in the livers of Card6-HKO mice but repressed in those of Card6-HTG mice. Furthermore, ASK1 knockdown normalized the effect of CARD6 deficiency on the activation of NF-κB, JNK and p38, while ASK1 overexpression abrogated the suppressive effect of CARD6. CARD6 was also shown to interact with ASK1. Mutant CARD6 that lacked the ability to interact with ASK1 could not inhibit ASK1 and failed to protect against hepatic I/R injury. CONCLUSIONS: CARD6 is a novel protective factor against hepatic I/R injury that suppresses inflammation and liver cell death by inhibiting the ASK1 signaling pathway. LAY SUMMARY: The protein CARD6 plays an important role during the process of liver blood flow restriction (ischemia) and restoration (reperfusion). By suppressing the activity of ASK1, CARD6 can protect against hepatocyte injury. Targeting CARD6 is a potential strategy for prevention and treatment of ischemia/reperfusion injury.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/fisiologia , Fígado/irrigação sanguínea , MAP Quinase Quinase Quinase 5/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Humanos , Inflamação/prevenção & controle , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
8.
J Am Heart Assoc ; 7(12)2018 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-29887521

RESUMO

BACKGROUND: Tollip, a well-established endogenous modulator of Toll-like receptor signaling, is involved in cardiovascular diseases. The aim of this study was to investigate the role of Tollip in neointima formation and its associated mechanisms. METHODS AND RESULTS: In this study, transient increases in Tollip expression were observed in platelet-derived growth factor-BB-treated vascular smooth muscle cells and following vascular injury in mice. We then applied loss-of-function and gain-of-function approaches to elucidate the effects of Tollip on neointima formation. While exaggerated neointima formation was observed in Tollip-deficient murine neointima formation models, Tollip overexpression alleviated vascular injury-induced neointima formation by preventing vascular smooth muscle cell proliferation, dedifferentiation, and migration. Mechanistically, we demonstrated that Tollip overexpression may exert a protective role in the vasculature by suppressing Akt-dependent signaling, which was further confirmed in rescue experiments using the Akt-specific inhibitor (AKTI). CONCLUSIONS: Our findings indicate that Tollip protects against neointima formation by negatively regulating vascular smooth muscle cell proliferation, dedifferentiation, and migration in an Akt-dependent manner. Upregulation of Tollip may be a promising strategy for treating vascular remodeling-related diseases.


Assuntos
Lesões das Artérias Carótidas/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Neointima , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Externa/enzimologia , Artéria Carótida Externa/patologia , Desdiferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Doença Arterial Periférica/enzimologia , Doença Arterial Periférica/patologia , Transdução de Sinais
9.
J Am Heart Assoc ; 7(13)2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29945911

RESUMO

BACKGROUND: Carboxyl-terminal modulator protein (CTMP) has been implicated in cancer, brain injury, and obesity. However, the role of CTMP in pathological cardiac hypertrophy has not been identified. METHODS AND RESULTS: In this study, decreased expression of CTMP was observed in both human failing hearts and murine hypertrophied hearts. To further explore the potential involvement of CTMP in pathological cardiac hypertrophy, cardiac-specific CTMP knockout and overexpression mice were generated. In vivo experiments revealed that CTMP deficiency exacerbated the cardiac hypertrophy, fibrosis, and function induced by pressure overload, whereas CTMP overexpression alleviated the response to hypertrophic stimuli. Consistent with the in vivo results, adenovirus-mediated gain-of-function or loss-of-function experiments showed that CTMP also exerted a protective effect against hypertrophic responses to angiotensin II in vitro. Mechanistically, CTMP ameliorated pathological cardiac hypertrophy through the blockade of the protein kinase B signaling pathway. Moreover, inhibition of protein kinase B activation with LY294002 rescued the deteriorated effect in aortic banding-treated cardiac-specific CTMP knockout mice. CONCLUSIONS: Taken together, these findings imply, for the first time, that increasing the cardiac expression of CTMP may be a novel therapeutic strategy for pathological cardiac hypertrophy.


Assuntos
Proteínas de Transporte/metabolismo , Hipertrofia Ventricular Esquerda/prevenção & controle , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Humanos , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Miócitos Cardíacos/patologia , Palmitoil-CoA Hidrolase , Ratos Sprague-Dawley , Transdução de Sinais , Tioléster Hidrolases/metabolismo
10.
Hepatology ; 68(5): 1786-1803, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29698567

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation, and the pathogenic mechanism of NAFLD is poorly understood. Ubiquitin-specific peptidase 10 (USP10), a member of the ubiquitin-specific protease family, is involved in environmental stress responses, tumor growth, inflammation, and cellular metabolism. However, the role of USP10 in hepatic steatosis, insulin resistance, and inflammation remains largely unexplored. USP10 expression was detected in livers of patients with NAFLD, mice with high-fat diet (HFD)-induced obesity, and genetically obese (ob/ob) mice, as well as in palmitate-induced hepatocytes. The function of USP10 in hepatic steatosis, insulin resistance, and inflammation was investigated using hepatocyte-specific USP10 deficiency or overexpression in mice induced by HFD treatment or genetic defect. The molecular mechanisms underlying USP10-regulated hepatic steatosis were further investigated in HFD-treated mice. USP10 expression was significantly decreased in the fatty livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. USP10 deficiency exacerbated the metabolic dysfunction induced by HFD treatment for 12 weeks. Conversely, USP10 overexpression significantly suppressed metabolic dysfunction in mice after HFD treatment and inhibited the development of NAFLD in ob/ob mice. Further investigation indicated that USP10 regulates hepatic steatosis by interacting with Sirt6 and inhibiting its ubiquitination and degradation. Sirt6 overexpression markedly ameliorated the effects of USP10 deficiency in hepatic steatosis, insulin resistance, and inflammation. Conversely, Sirt6 deficiency decreased the ameliorative effects of USP10 overexpression in response to HFD treatment. Conclusion: USP10 inhibits hepatic steatosis, insulin resistance, and inflammation through Sirt6.


Assuntos
Hepatopatia Gordurosa não Alcoólica/metabolismo , Sirtuínas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Western Blotting , Técnicas de Cultura de Células , Citocinas/sangue , Humanos , Imunoprecipitação/métodos , Resistência à Insulina/genética , Lipídeos , Fígado/metabolismo , Fígado/patologia , Testes de Função Hepática/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
11.
Nat Med ; 24(2): 213-223, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29291351

RESUMO

Nonalcoholic steatohepatitis (NASH) is a common clinical condition that can lead to advanced liver diseases. Lack of effective pharmacotherapies for NASH is largely attributable to an incomplete understanding of its pathogenesis. The deubiquitinase cylindromatosis (CYLD) plays key roles in inflammation and cancer. Here we identified CYLD as a suppressor of NASH in mice and in monkeys. CYLD is progressively degraded upon interaction with the E3 ligase TRIM47 in proportion to NASH severity. We observed that overexpression of Cyld in hepatocytes concomitantly inhibits lipid accumulation, insulin resistance, inflammation and fibrosis in mice with NASH induced in an experimental setting. Mechanistically, CYLD interacts directly with the kinase TAK1 and removes its K63-linked polyubiquitin chain, which blocks downstream activation of the JNK-p38 cascades. Notably, reconstitution of hepatic CYLD expression effectively reverses disease progression in mice with dietary or genetically induced NASH and in high-fat diet-fed monkeys predisposed to metabolic syndrome. Collectively, our findings demonstrate that CYLD mitigates NASH severity and identify the CYLD-TAK1 axis as a promising therapeutic target for management of the disease.


Assuntos
Cisteína Endopeptidases/genética , Inflamação/genética , MAP Quinase Quinase Quinases/genética , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Proteínas de Transporte/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Enzima Desubiquitinante CYLD , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Haplorrinos , Humanos , Inflamação/fisiopatologia , Fígado/metabolismo , Fígado/patologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Camundongos , Proteínas de Neoplasias/genética , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Proteínas Nucleares/genética , Ligação Proteica/genética , Índice de Gravidade de Doença , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
12.
Circulation ; 137(14): 1486-1504, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29229612

RESUMO

BACKGROUND: Cardiac hypertrophy and its resultant heart failure are among the most common causes of mortality worldwide. Abnormal protein degradation, especially the impaired lysosomal degradation of large organelles and membrane proteins, is involved in the progression of cardiac hypertrophy. However, the underlying mechanisms have not been fully elucidated. METHODS: We investigated cardiac transmembrane BAX inhibitor motif containing 1 (TMBIM1) mRNA and protein expression levels in samples from patients with heart failure and mice with aortic banding (AB)-induced cardiac hypertrophy. We generated cardiac-specific Tmbim1 knockout mice and cardiac-specific Tmbim1-overexpressing transgenic mice and then challenged them with AB surgery. We used microarray, confocal image, and coimmunoprecipitation analyses to identify the downstream targets of TMBIM1 in cardiac hypertrophy. Tmbim1/Tlr4 double-knockout mice were generated to investigate whether the effects of TMBIM1 on cardiac hypertrophy were Toll-like receptor 4 (TLR4) dependent. Finally, lentivirus-mediated TMBIM1 overexpression in a monkey AB model was performed to evaluate the therapeutic potential of TMBIM1. RESULTS: TMBIM1 expression was significantly downregulated on hypertrophic stimuli in both human and mice heart samples. Silencing cardiac Tmbim1 aggravated AB-induced cardiac hypertrophy. This effect was blunted by Tmbim1 overexpression. Transcriptome profiling revealed that the TLR4 signaling pathway was disrupted dramatically by manipulation of Tmbim1. The effects of TMBIM1 on cardiac hypertrophy were shown to be dependent on TLR4 in double-knockout mice. Fluorescent staining indicated that TMBIM1 promoted the lysosome-mediated degradation of activated TLR4. Coimmunoprecipitation assays confirmed that TMBIM1 directly interacted with tumor susceptibility gene 101 via a PTAP motif and accelerated the formation of multivesicular bodies that delivered TLR4 to the lysosomes. Finally, lentivirus-mediated TMBIM1 overexpression reversed AB-induced cardiac hypertrophy in monkeys. CONCLUSIONS: TMBIM1 protects against pathological cardiac hypertrophy through promoting the lysosomal degradation of activated TLR4. Our findings reveal the central role of TMBIM1 as a multivesicular body regulator in the progression of pathological cardiac hypertrophy, as well as the role of vesicle trafficking in signaling regulation during cardiac hypertrophy. Moreover, targeting TMBIM1 could be a novel therapeutic strategy for treating cardiac hypertrophy and heart failure.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cardiomegalia/patologia , Insuficiência Cardíaca/patologia , Motivos de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Haplorrinos , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
Nat Med ; 24(1): 73-83, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29227475

RESUMO

Hepatic ischemia-reperfusion (IR) injury is a common clinical issue lacking effective therapy and validated pharmacological targets. Here, using integrative 'omics' analysis, we identified an arachidonate 12-lipoxygenase (ALOX12)-12-hydroxyeicosatetraenoic acid (12-HETE)-G-protein-coupled receptor 31 (GPR31) signaling axis as a key determinant of the hepatic IR process. We found that ALOX12 was markedly upregulated in hepatocytes during ischemia to promote 12-HETE accumulation and that 12-HETE then directly binds to GPR31, triggering an inflammatory response that exacerbates liver damage. Notably, blocking 12-HETE production inhibits IR-induced liver dysfunction, inflammation and cell death in mice and pigs. Furthermore, we established a nonhuman primate hepatic IR model that closely recapitulates clinical liver dysfunction following liver resection. Most strikingly, blocking 12-HETE accumulation effectively attenuated all pathologies of hepatic IR in this model. Collectively, this study has revealed previously uncharacterized metabolic reprogramming involving an ALOX12-12-HETE-GPR31 axis that functionally determines hepatic IR procession. We have also provided proof of concept that blocking 12-HETE production is a promising strategy for preventing and treating IR-induced liver damage.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Fígado/irrigação sanguínea , Receptores Acoplados a Proteínas G/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inibidores , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Metabolismo dos Lipídeos , Camundongos , Traumatismo por Reperfusão/parasitologia , Suínos
14.
Nat Med ; 24(1): 84-94, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29227477

RESUMO

Activation of apoptosis signal-regulating kinase 1 (ASK1) in hepatocytes is a key process in the progression of nonalcoholic steatohepatitis (NASH) and a promising target for treatment of the condition. However, the mechanism underlying ASK1 activation is still unclear, and thus the endogenous regulators of this kinase remain open to be exploited as potential therapeutic targets. In screening for proteins that interact with ASK1 in the context of NASH, we identified the deubiquitinase tumor necrosis factor alpha-induced protein 3 (TNFAIP3) as a key endogenous suppressor of ASK1 activation, and we found that TNFAIP3 directly interacts with and deubiquitinates ASK1 in hepatocytes. Hepatocyte-specific ablation of Tnfaip3 exacerbated nonalcoholic fatty liver disease- and NASH-related phenotypes in mice, including glucose metabolism disorders, lipid accumulation and enhanced inflammation, in an ASK1-dependent manner. In contrast, transgenic or adeno-associated virus-mediated TNFAIP3 gene delivery in the liver in both mouse and nonhuman primate models of NASH substantially blocked the onset and progression of the disease. These results implicate TNFAIP3 as a functionally important endogenous suppressor of ASK1 hyperactivation in the pathogenesis of NASH and identify it as a potential new molecular target for NASH therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fígado/enzimologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Apoptose , Dieta Hiperlipídica , Fibrose/prevenção & controle , Humanos , Inflamação/prevenção & controle , Resistência à Insulina , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Transdução de Sinais , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Hepatology ; 67(4): 1320-1338, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29077210

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a prevalent and complex disease that confers a high risk of severe liver disorders. Despite such public and clinical health importance, very few effective therapies are currently available for NAFLD. We report a protective function and the underlying mechanism of dual-specificity phosphatase 14 (DUSP14) in NAFLD and related metabolic disorders. Insulin resistance, hepatic lipid accumulation, and concomitant inflammatory responses, key pathological processes involved in NAFLD development, were significantly ameliorated by hepatocyte-specific DUSP14 overexpression (DUSP14-HTG) in high-fat diet (HFD)-induced or genetically obese mouse models. By contrast, specific DUSP14 deficiency in hepatocytes (DUSP14-HKO) aggravated these pathological alterations. We provided mechanistic evidence that DUSP14 directly binds to and dephosphorylates transforming growth factor ß-activated kinase 1 (TAK1), resulting in the reduced activation of TAK1 and its downstream signaling molecules c-Jun N-terminal kinase 1 (JNK), p38, and nuclear factor kappa B NF-κB. This effect was further evidenced by the finding that inhibiting TAK1 activity effectively attenuated the deterioration of glucolipid metabolic phenotype in DUSP14-HKO mice challenged by HFD administration. Furthermore, we identified that both the binding domain and the phosphatase activity of DUSP14 are required for its protective role against hepatic steatosis, because interruption of the DUSP14-TAK1 interaction abolished the mitigative effects of DUSP14. CONCLUSION: Hepatocyte DUSP14 is required for maintaining hepatic metabolic homeostasis and for suppressing inflammation, a novel function that relies on constraining TAK1 hyperactivation. (Hepatology 2018;67:1320-1338).


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Hepatócitos/metabolismo , Homeostase/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Resistência à Insulina/genética , Fígado/metabolismo , Fígado/patologia , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
16.
J Neurosci ; 37(50): 12123-12140, 2017 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-29114077

RESUMO

Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury.SIGNIFICANCE STATEMENT Stroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting the TRAF6-Rac1 axis may provide a novel therapeutic strategy for stroke recovery.


Assuntos
Infarto da Artéria Cerebral Média/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Traumatismo por Reperfusão/enzimologia , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Estresse Oxidativo , RNA Interferente Pequeno/farmacologia , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/genética , Transfecção , Ubiquitinação , Regulação para Cima
18.
Circulation ; 136(15): 1412-1433, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-28851732

RESUMO

BACKGROUND: The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown. METHODS: IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)-specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)-TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated. RESULTS: IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation. CONCLUSIONS: IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.


Assuntos
Regulação da Expressão Gênica , Fatores Reguladores de Interferon , Fatores de Transcrição Kruppel-Like , Músculo Liso Vascular , Neointima , Animais , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neointima/genética , Neointima/metabolismo , Neointima/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Ratos
19.
Hypertension ; 70(4): 770-779, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28827473

RESUMO

Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting Mfge8 in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific Mfge8 overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either Mfge8-knockout or Mfge8-overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3ß (glycogen synthase kinase-3ß)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by Mfge8 deficiency but suppressed by Mfge8 overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of Mfge8 deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy.


Assuntos
Antígenos de Superfície , Cardiomegalia , Insuficiência Cardíaca , Proteínas do Leite , Remodelação Ventricular/fisiologia , Angiotensina II/metabolismo , Animais , Antígenos de Superfície/sangue , Antígenos de Superfície/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Cardiomegalia/complicações , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Reprogramação Celular/fisiologia , Regulação para Baixo/fisiologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos , Proteínas do Leite/sangue , Proteínas do Leite/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Índice de Gravidade de Doença , Transdução de Sinais/fisiologia , Estatística como Assunto
20.
Clin Sci (Lond) ; 131(17): 2275-2288, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28743735

RESUMO

Atherosclerosis is a chronic inflammatory disease. LILRB4 is associated with the pathological processes of various inflammatory diseases. However, the potential function and underlying mechanisms of LILRB4 in atherogenesis remain to be investigated. In this study, LILRB4 expression was examined in both human and mouse atherosclerotic plaques. The effects and possible mechanisms of LILRB4 in atherogenesis and plaque instability were evaluated in LILRB4-/-ApoE-/- and ApoE-/- mice fed a high-fat diet. We found that LILRB4 was located primarily in macrophages, and its expression was up-regulated in atherosclerotic lesions from human coronary arteries and mouse aortic roots. LILRB4 deficiency significantly accelerated the development of atherosclerotic lesions and increased the instability of plaques, as evidenced by the increased infiltration of lipids, decreased amount of collagen components and smooth muscle cells. Moreover, LILRB4 deficiency in bone marrow-derived cells promoted the development of atherosclerosis. In vivo and in vitro analyses revealed that the pro-inflammatory effects of LILRB4 deficiency were mediated by the increased activation of NF-κB signaling due to decreased Shp1 phosphorylation. In conclusion, the present study indicates that LILRB4 deficiency promotes atherogenesis, at least partly, through reduced Shp1 phosphorylation, which subsequently enhances the NF-κB-mediated inflammatory response. Thus, targeting the "LILRB4-Shp1" axis may be a novel therapeutic approach for atherosclerosis.

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