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1.
PLoS One ; 15(8): e0234492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790760

RESUMO

Endothelial injury is a common manifestation in IgA nephropathy (IgAN). After the previous identification of the upregulated soluble fms-like tyrosine kinase-1 (sFlt-1) correlated with endothelial injury in IgAN, in the present study, we further explored the role of sFlt-1 in endothelial injury in IgAN. We enrolled 72 patients with IgAN and detected the sFlt-1 levels. The polymeric IgA1 (pIgA1) complexes were isolated from the pooled plasma samples of another 10 patients with IgAN. Apoptosis proteins were detected in cultured human umbilical vein endothelial cells (HUVECs) with the stimulation of recombinant sFlt-1 or the caspase-9 inhibitor Z-LEHD-FMK. We identified there were positive correlations between sFlt-1 and IgA-IgG complex as well as vWF levels in patients with IgAN. The sFlt-1 levels in HUVECs were significantly upregulated by pIgA1 complex derived from IgAN patients in a concentration-dependent manner. The proliferation ability of HUVECs was damaged when stimulated with sFlt-1 protein in a time- and dose- dependent manner. And the apoptosis rate was up-regulated significantly as the stimulation concentrations of sFlt-1 increased. We found sFlt-1 challenge could significantly increase the expression of vWF. In addition, sFlt-1 increased the levels of caspase-9, caspase-3, Bax and mitochondrial membrane potential; facilitated the release of cytochrome C from mitochondria to cytoplasma. In contrast, Z-LEHD-FMK attenuated high sFlt-1-induced HUVECs apoptosis. In conclusion, our study demonstrated that sFlt-1 expression was up-regulated by the challenge of pIgA1 complex derived from patients with IgAN. Furthermore, increased sFlt-1 facilitated human umbilical vein endothelial cells apoptosis via the mitochondrial-dependent pathway.


Assuntos
Endotélio Vascular/fisiopatologia , Glomerulonefrite por IGA/fisiopatologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Adulto , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Caspase 9/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oligopeptídeos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Adulto Jovem
2.
Cell Immunol ; 349: 104046, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32057354

RESUMO

Uncontrolled activation of NLRP3 inflammasome initiates a series of human inflammatory diseases. Targeting NLRP3 inflammasome has attracted considerable attention in developing potential therapeutic interventions. Here, we reported that dehydrocostus lactone (DCL), a main component of Saussurea lappa from the traditional Chinese medicine, inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent interleukin (IL)-1ß production in primary mouse macrophages and human peripheral blood mononuclear cells and exerted an inhibitory effect on NLRP3-driven inflammation. Mechanistically, DCL significantly blocked the ASC oligomerization, which is essential for the assembly of activated inflammasome. Importantly, in vivo experiments showed that DCL reduced IL-1ß secretion and peritoneal neutrophils recruitment in LPS-mediated inflammation mouse model, which is demonstrated to be NLRP3 dependent. These results suggest that DCL is a potent pharmacological inhibitor of NLRP3 inflammasome and may be developed as a therapeutic drug for treating NLRP3-associated diseases.


Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamação/prevenção & controle , Lactonas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sesquiterpenos/farmacologia , Adulto , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/fisiologia , Caspase 1/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/fisiologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Interleucina-1beta/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Nigericina/farmacologia , Poli I-C/farmacologia , Polimerização/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Ácido Úrico/farmacologia
3.
Mol Cell ; 77(6): 1222-1236.e13, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32048998

RESUMO

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Exorribonucleases/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Biossíntese de Proteínas , RNA Helicases/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fases de Leitura Aberta , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/genética , Ribossomos/metabolismo
4.
Transplantation ; 104(9): 1869-1878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32058468

RESUMO

BACKGROUND: Triple progressive thermopreconditioning (3PTP) may induce high Hsp-70 expression to maintain cardiac function. We suggest that 3PTP may reduce myocardial ischemia/reperfusion (I/R) injury during organ transplantation through Bag3/Hsp-70 mediated defense mechanisms. METHODS: Male Wistar rats were divided into sham control group and 72 h after 3PTP in a 42°C water bath (3PTP) group. Rats underwent 60 min of ischemia by occlusion of the left anterior descending coronary artery followed by 240 min reperfusion. Hemodynamic parameters, including the electrocardiogram, microcirculation, heart rate, left ventricular end-diastolic pressure, maximal rate of rise (+dp/dt), and fall (-dp/dt) in the left ventricular pressure for index of contraction and relaxation were determined. Myocardial infarct size was evaluated by the Evans blue-2,3,5-triphenyltetrazolium chloride method. 3PTP-induced protective mechanisms were determined by Western blot and immunohistochemistry. RESULTS: Cardiac I/R depressed cardiac microcirculation, induced S-T segment elevation, and R-R and P-R interval elongation increased infarct size associated with erythrocyte extravasation, leukocytes and macrophage/monocyte infiltration, granulocyte colony-stimulating factor, poly(ADP-ribose) polymerase 1 stain, and transferase-mediated dUTP-biotin nick end labeling positive cells. However, 3PTP evoked significant cardioprotection against I/R injury, characterized by the increased +dp/dt value and the decreased elevated left ventricular end-diastolic pressure, erythrocyte extravasation, leukocyte and macrophage/monocyte infiltration, granulocyte colony-stimulating factor expression, poly(ADP-ribose) polymerase 1 expression, transferase-mediated dUTP-biotin nick end labeling positive cells, and fragmentation and infarct area. In addition, 3PTP increased Hsp-70 and Bag3 expression and decreased Bax/Bcl-2 ratio, but did not affect the Beclin-1 and LC3-II/LC3-I ratio in the heart with I/R injury. CONCLUSIONS: 3PTP therapies may through Bag3 upregulation alleviate I/R injury-induced left ventricular structural deterioration and dysfunction.


Assuntos
Ventrículos do Coração/patologia , Precondicionamento Isquêmico Miocárdico , Contração Miocárdica/fisiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Disfunção Ventricular Esquerda/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Masculino , Microcirculação , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Ratos Wistar
5.
Life Sci ; 245: 117347, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981628

RESUMO

AIM: Oxidative stress plays an important role in myocardial ischemia-reperfusion injury. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was first identified in apoptosis induced by T cell receptor activation, and was shown to play a different role in different cell types and under different stimuli. The role and mechanism of PHLDA1 in oxidative stress-induced cardiomyocyte injury and cardiac ischemia-reperfusion were therefore determined. MAIN METHODS: Cell viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Mitochondrial membrane potential was measured using JC-1 test kit. Reactive oxygen species (ROS) production was detected using ROS kit. HE staining was used to detect histological morphology, 2,3,5-triphenyltetrazolium chloride staining to detect infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling staining to detect the apoptotic rate, and immunohistochemistry and western blot analysis to detect protein expression. The binding of PHLDA1 to Bcl-2 associated X (Bax) was detected by immunoprecipitation. KEY FINDINGS: The results indicated that PHLDA1 is highly expressed in oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. PHLDA1 overexpression in cardiomyocytes promoted oxidative stress-induced cardiomyocyte injury. At the same time, PHLDA1 knockdown improved oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. In addition, PHLDA1 binds to Bax and the interaction is enhanced under H2O2 stimulation. SIGNIFICANCE: The present results indicated that PHLDA1 interacts with Bax to participate in oxidative stress-induced cardiomyocyte injury and myocardial ischemia reperfusion injury.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Linhagem Celular , Citometria de Fluxo , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Masculino , Potencial da Membrana Mitocondrial , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
Mol Cell ; 77(3): 600-617.e4, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31952989

RESUMO

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or ß-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.


Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , Termogênese/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Dieta , Metabolismo Energético , Glucose/metabolismo , Glicólise/fisiologia , Células HEK293 , Humanos , Resistência à Insulina , Gotículas Lipídicas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NAD/fisiologia , NADH NADPH Oxirredutases/metabolismo , Obesidade/metabolismo , Oxirredução , Consumo de Oxigênio , Proteína Desacopladora 1/metabolismo
7.
Life Sci ; 242: 117159, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31837334

RESUMO

AIMS: It has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models. MAIN METHODS: Intraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit. KEY FINDINGS: The protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats. SIGNIFICANCE: Siah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Monocrotalina/farmacologia , Proteínas Nucleares/fisiologia , Hipertensão Arterial Pulmonar/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Remodelação Vascular/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Immunoblotting , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Hipertensão Arterial Pulmonar/induzido quimicamente , Hipertensão Arterial Pulmonar/fisiopatologia , Ratos , Ratos Sprague-Dawley , Remodelação Vascular/fisiologia
8.
J Zhejiang Univ Sci B ; 20(11): 928-932, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31595729

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and is the second leading cause of cancer mortality with an estimated 745 500 deaths annually (Jemal et al., 2011). Although new therapeutic modalities including novel chemotherapeutic interventions and targeted therapy have been applied, the prognosis of HCC patients remains unsatisfactory due to the high incidence of intrahepatic and distal metastases (Siegel et al., 2018).


Assuntos
Carcinoma Hepatocelular/genética , Genoma , Neoplasias Hepáticas/genética , MicroRNAs/análise , Proteínas Reguladoras de Apoptose/fisiologia , Biomarcadores , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Hipóxia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Estadiamento de Neoplasias , Prognóstico , Proteínas Repressoras/fisiologia
9.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(4): 536-540, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31484618

RESUMO

Apoptosis plays important roles in maintaining normal development and homeostasis and in the pathophysiological processes of various diseases.Previous studies have shown that cardiomyocyte apoptosis is involved in the development of various cardiovascular diseases.The apoptosis repressor with caspase recruitment domain(ARC)is abundantly expressed in heart,which makes ARC a unique and central cardioprotective factor via anti-apoptotic pathway.This article reviews the structure and characteristics of ARC as well as the roles and mechanisms of ARC in cardiovascular diseases.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Doenças Cardiovasculares , Domínio de Ativação e Recrutamento de Caspases , Humanos
11.
PLoS Pathog ; 15(8): e1008004, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412082

RESUMO

Fas-associated factor 1 is a death-promoting protein that induces apoptosis by interacting with the Fas receptor. Until now, FAF1 was reported to interact potentially with diverse proteins and to function as a negative and/or positive regulator of several cellular possesses. However, the role of FAF1 in defense against bacterial infection remains unclear. Here, we show that FAF1 plays a pivotal role in activating NADPH oxidase in macrophages during Listeria monocytogenes infection. Upon infection by L. monocytogenes, FAF1 interacts with p67phox (an activator of the NADPH oxidase complex), thereby facilitating its stabilization and increasing the activity of NADPH oxidase. Consequently, knockdown or ectopic expression of FAF1 had a marked effect on production of ROS, proinflammatory cytokines, and antibacterial activity, in macrophages upon stimulation of TLR2 or after infection with L. monocytogenes. Consistent with this, FAF1gt/gt mice, which are knocked down in FAF1, showed weaker inflammatory responses than wild-type mice; these weaker responses led to increased replication of L. monocytogenes. Collectively, these findings suggest that FAF1 positively regulates NADPH oxidase-mediated ROS production and antibacterial defenses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Imunidade Inata/imunologia , Inflamação/imunologia , Listeriose/imunologia , Macrófagos/imunologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/microbiologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Listeriose/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais
12.
J Biomed Sci ; 26(1): 43, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31167655

RESUMO

BACKGROUND: Stroke is the second most common cause of deaths worldwide. After an ischemic stroke, the proliferated reactive astrocytes in the peri-infarct areas play a beneficial role in neuronal survival. As such, astrocytes have gradually become a target for neuroprotection in stroke. The present study assessed the hypothesis that Pinin (Pnn), originally identified as a nuclear and desmosome-associated protein and is now known to possess anti-apoptotic capacity, protects astrocytes from cell death after ischemic stroke and delineated the underlying mechanisms. METHODS: In in vivo experiments, adult male Sprague-Dawley rats (12-week old) were used to induce acute focal cerebral ischemia employing the middle cerebral artery occlusion (MCAO) method. In in vitro experiments, postnatal day 1 (P1) Sprague-Dawley rat pups were used to prepare cultures of primary astrocytes. Oxygen-glucose deprivation (OGD) and re-oxygenation (OGD/R) procedures were employed to mimic the hypoxic-ischemic condition of stroke in those astrocytes. RESULTS: We found in the peri-infarct area of the ipsilateral cortex and striatum in Sprague-Dawley rats after transient MCAO an increase in Pnn expression that correlated positively with the time-course of infarction as detected by T2-weighted imaging and triphenyltetrazolium chloride staining, augmented number of reactive astrocytes that double-labelled with Pnn as determined by immunofluorescence, and enhanced cytotoxic edema as revealed by diffusion weighted imaging; but mirrored the decreased cleaved caspase-3 as measured by western blot. In an OGD and OGD/R model using primary cultured astrocytes, treatment with Pnn siRNA doubled the chance of surviving astrocytes to manifest cell death as revealed by flow cytometry, and blunted activated ERK signaling, reduced Bcl-2 expression and augmented cleaved caspase 3 detected by western blot in the normoxia, OGD or OGD/R group. Gene-knockdown of Pnn also impeded the reversal from decline in cell viability, elevation in lactate dehydrogenase leakage and decrease in ATP production in the OGD/R group. CONCLUSION: We conclude that the endogenous Pnn participates in neuroprotection after acute ischemic stroke by preserving the viability of astrocytes that survived the ischemic challenge via maintenance of mitochondrial anti-apoptotic and bioenergetics functions.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Astrócitos/patologia , Isquemia Encefálica/patologia , Moléculas de Adesão Celular/fisiologia , Mitocôndrias/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Moléculas de Adesão Celular/genética , Morte Celular/genética , Morte Celular/fisiologia , Sobrevivência Celular , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
13.
EMBO J ; 38(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30858281

RESUMO

SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Esteróis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
14.
Arterioscler Thromb Vasc Biol ; 39(3): 402-412, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30602303

RESUMO

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Repressoras/fisiologia , Tromboplastina/biossíntese , Trombose/prevenção & controle , Animais , Antitrombina III/análise , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Transplante de Medula Óssea , Células Cultivadas , Cloretos/toxicidade , Imunoprecipitação da Cromatina , Regulação para Baixo , Feminino , Compostos Férricos/toxicidade , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Hidrolases/análise , Agregação Plaquetária , Interferência de RNA , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Tromboplastina/genética , Trombose/induzido quimicamente , Transcrição Genética
16.
Int Immunopharmacol ; 68: 145-155, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30634142

RESUMO

Gasdermin D (GSDMD), a genetic substrate for inflammatory caspases, plays a central role in pyroptosis of macrophages and release of interleukin­1ß (IL-1ß), but was mainly referred to microbial infection. High mobility group box-1 (HMGB1), served as an alarm molecule during various pathological process, has been widely recognized to be involved in liver ischemia-reperfusion (I/R). Glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, was recently referred to have ability to prevent I/R induced liver injury by inhibiting HMGB1 expression and activity. However, the mechanisms responsible for damage amelioration subsequently to HMGB1 inhibition during liver I/R remain enigmatic. This study was designed to explore the functional role and molecular mechanism of glycyrrhizin in the regulation of I/R induced liver injury. We found that liver I/R promotes GSDMD-mediated pyroptotic cell death of Kupffer cells, which was inhibited by glycyrrhizin. Interestingly, endogenous HMGB1, not exogenous one, was involved in hypoxia-reoxygenation (H/R) induced pyroptosis. Moreover, GSDMD knockdown protects kupffer cells against H/R induced pyroptosis in vitro. Here, we report, for the first time, that glycyrrhizin attenuated tissue damage and kupffer cells pyroptosis during liver ischemia-reperfusion injury (LIRI) and identify a previously unrecognized HMGB1- dependent GSDMD- mediated signaling pathway in the mechanism of kupffer cells pyroptosis induced by H/R. Our findings provide the first demonstration of GSDMD-determined pyroptotic cell death responsible for I/R induced release of IL-1ß and this would provide a mandate to better understand the unconventional mechanisms of cytokine release in the sterile innate immune system.


Assuntos
Anti-Inflamatórios , Proteínas Reguladoras de Apoptose/fisiologia , Ácido Glicirrízico , Proteína HMGB1/metabolismo , Macrófagos do Fígado/efeitos dos fármacos , Hepatopatias , Piroptose/efeitos dos fármacos , Traumatismo por Reperfusão , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/uso terapêutico , Interleucina-1beta/metabolismo , Macrófagos do Fígado/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
17.
PLoS One ; 14(1): e0210526, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30629682

RESUMO

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Sistemas CRISPR-Cas , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Dano ao DNA , Reparo do DNA , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo
18.
Life Sci ; 219: 129-135, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30639391

RESUMO

AIMS: Aberrantly expressed miRNAs are demonstrated to be involved in the development of congenital heart disease (CHD). miR-9 was proposed to be upregulated in cardiac tissues from CHD cases. However, the role of miR-9 in hypoxia-induced cardiomyocytes and the potential mechanism are far from being addressed. MAIN METHODS: qRT-PCR and western blot analysis were performed to detect miR-9 and Yes-associated protein 1 (Yap1) expressions in hypoxic H9c2 cells. CCK-8, flow cytometry analysis, caspase-3/7 activity assay were applied to evaluate cell proliferation, apoptosis, and caspase-3/7 activity, respectively. The interaction between miR-9 and Yap1 was explored by luciferase reporter assay, qRT-PCR and western blot. KEY FINDINGS: miR-9 was upregulated and Yap1 was downregulated in H9c2 cells in response to hypoxia in a time-dependent manner. Knockdown of miR-9 promoted cell proliferation, and inhibited apoptosis and caspase-3/7 activity in hypoxic H9c2 cells, while miR-9 overexpression exerted the opposite effects on hypoxic H9c2 cells. In addition, Yap1 was a direct target of miR-9 in H9c2 cells. Yap1 knockdown suppressed cell proliferation and promoted apoptosis in hypoxia-exposed H9c2 cells. Yap1 knockdown attenuated the effect of anti-miR-9 on cell proliferation and apoptosis in hypoxia-exposed H9c2 cells. SIGNIFICANCE: miR-9 knockdown inhibited hypoxia-induced cardiomyocyte apoptosis by targeting Yap1. Our study provided a novel insight into the mechanism of the adaptation of cardiomyocytes to chronic hypoxia.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Hipóxia/fisiopatologia , MicroRNAs/fisiologia , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas c-yes/fisiologia , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Proliferação de Células , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real
19.
Blood ; 133(3): 193-204, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30442679

RESUMO

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1 ECKO ) or Pdcd10 (Pdcd10 ECKO ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10 ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10 ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.


Assuntos
Anticoagulantes/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Hemorragia Cerebral/diagnóstico , Endotélio Vascular/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/complicações , Proteína KRIT1/fisiologia , Proteínas de Membrana/fisiologia , Proteína C/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Trombomodulina/sangue , Adulto , Animais , Coagulação Sanguínea , Estudos de Casos e Controles , Hemorragia Cerebral/sangue , Hemorragia Cerebral/etiologia , Receptor de Proteína C Endotelial/metabolismo , Endotélio Vascular/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Adulto Jovem
20.
Behav Brain Res ; 356: 8-17, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30092249

RESUMO

Ischemia/reperfusion (I/R) injuries commonly lead to breakdown of the blood-brain barrier (BBB). Restoration of the BBB can relieve neurologic damage caused by I/R injuries. The Hippo/YAP signaling pathway mediates cell proliferation, regulated cell death, and differentiation in various organisms and has been shown to participate in the restoration of the heart after I/R. In this study, we investigated whether the Hippo/YAP pathway plays a role in I/R injury in brain, especially in regard to I/R-induced BBB breakdown. The results of our study indicate that I/R injury led to an overall decrease in activity of the core proteins, YAP and TAZ, over a 24-h period. The most dramatic change was observed 1.5 h after reperfusion. In rats that underwent 1.5 h of reperfusion, intraperitoneal injection of YAP agonist dexamethasone activated YAP and TAZ and led to improved neurologic function, smaller brain infarct sizes, increased levels of tight junction proteins, decreased BBB permeability, decreased cerebral edema, and less apoptosis. Our results suggest that YAP exerts neuroprotective effects on the damaged brain that are likely related to restoration of the BBB.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Fatores de Transcrição/metabolismo , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/fisiopatologia , Isquemia Encefálica/fisiopatologia , Dexametasona/farmacologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Fármacos Neuroprotetores/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/fisiologia
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