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1.
Circ Res ; 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34702049

RESUMO

Rationale: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. Objective: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. Methods and Results: Using SMCs, mouse models, and human atherosclerosis specimens, we found that focal adhesion kinase (FAK) activation elicits SMC dedifferentiation by stabilizing DNA methyltransferase 3A (DNMT3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared to vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. Conclusions: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

2.
Nat Commun ; 12(1): 5551, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548489

RESUMO

While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors.


Assuntos
Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Glioblastoma/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas do Tecido Nervoso/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Proteínas Repressoras/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Éxons , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Xenoenxertos , Humanos , Íntrons , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073834

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid ß-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos/metabolismo , Lipogênese/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Acetil-CoA Carboxilase/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica , Ácido Graxo Sintases/metabolismo , Insulina/sangue , Resistência à Insulina/fisiologia , Lipase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Obesidade/metabolismo , Receptores Purinérgicos P2Y2/deficiência , Receptores Purinérgicos P2Y2/genética , Estearoil-CoA Dessaturase/metabolismo
4.
Cardiovasc Res ; 2021 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-33839758

RESUMO

AIM: Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood. METHODS AND RESULTS: We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets the CDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator, fizzy-related 1 (Fzr1) in the nucleus, which promotes ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signaling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enables proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activates FAK in the cytosol and increased Skp2 and decreased p27 and p21 levels. CONCLUSIONS: Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signaling negatively regulates CDK activity in VSMC proliferation. TRANSLATIONAL PERSPECTIVE: Increased VSMC proliferation contributes to pathological vessel narrowing in atherosclerosisand following vascular interventions. Blocking VSMC proliferation will reduce atherosclerosisprogression and increase patency of vascular interventions. We found that forced nuclear FAKlocalization by FAK inhibition reduced VSMC proliferation upon vessel injury. Nuclear FAKdecreased Skp2 protein expression by proteasomal degradation, thereby increasing theexpression of cell cycle inhibitors p27 and p21 and blocking cell cycle progression. This studyhas demonstrated the potential for FAK inhibitors in blocking VSMC proliferation to treat vessel narrowing diseases.

5.
Inflammation ; 44(3): 1130-1144, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33527321

RESUMO

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.

6.
Int J Mol Sci ; 22(4)2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33562139

RESUMO

Diabetic nephropathy (DN) is a common pathological feature in patients with diabetes and the leading cause of end-stage renal disease. Although several pharmacological agents have been developed, the management of DN remains challenging. Geniposide, a natural compound has been reported for anti-inflammatory and anti-diabetic effects; however, its role in DN remains poorly understood. This study investigated the protective effects of geniposide on DN and its underlying mechanisms. We used a C57BL/6 mouse model of DN in combination with a high-fat diet and streptozotocin after unilateral nephrectomy and treated with geniposide by oral gavage for 5 weeks. Geniposide effectively improves DN-induced renal structural and functional abnormalities by reducing albuminuria, podocyte loss, glomerular and tubular injury, renal inflammation and interstitial fibrosis. These changes induced by geniposide were associated with an increase of AMPK activity to enhance ULK1-mediated autophagy response and a decrease of AKT activity to block oxidative stress, inflammation and fibrosis in diabetic kidney. In addition, geniposide increased the activities of PKA and GSK3ß, possibly modulating AMPK and AKT pathways, efficiently improving renal dysfunction and ameliorating the progression of DN. Conclusively, geniposide enhances ULK1-mediated autophagy and reduces oxidative stress, inflammation and fibrosis, suggesting geniposide as a promising treatment for DN.


Assuntos
Anti-Inflamatórios/farmacologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Iridoides/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/prevenção & controle , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos
7.
Am J Chin Med ; 49(1): 131-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33371811

RESUMO

Alcoholic liver damage is caused by ethanol and its oxidized intermediates, and endotoxin-induced acute liver failure is mediated by apoptosis and inflammation. We investigated whether extracts of sprouts of Panax ginseng (SG) attenuate alcohol or endotoxin-induced acute liver injury in mice. Whole SG contains eight times more ginsenosides than the root and, because it grows quickly ([Formula: see text]30 days) without using pesticides, the whole-plant can be harvested. The extracts were enriched in phenolics and flavonoids and showed high radical scavenging activities. Mice received oral administration of SG or fermented SG (FSG) extracts 1 h before an injection of either ethanol or lipopolysaccharide and D-galactosamine (LPS/GalN). The latency of righting reflex was monitored to examine the effect of extracts on relieving hangover symptoms. The results indicate that FSG significantly reduced the latency of righting reflex, SG and FSG increased the activity and expression of ethanol-metabolizing enzymes, and FSG decreased hepatic necrosis and plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). During the ethanol metabolism, cytochrome P450 2E1 expression was increased, but 4-hydroxynonenal levels were decreased by the extracts due to their anti-oxidant activity. LPS/GalN-induced liver injury was reduced by SG and FSG; plasma ALT and AST levels, hepatic necrosis, and apoptotic and inflammatory markers were all decreased. In conclusion, SG extracts attenuated ethanol-induced hangover and endotoxin-induced acute liver injury, and fermentation enhanced the efficacy with regard to relieving hangover.


Assuntos
Intoxicação Alcoólica/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Fermentação , Flavonoides/análise , Panax/química , Fenóis/análise , Fitoterapia , Plântula/química , Administração Oral , Animais , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Sequestradores de Radicais Livres , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR
8.
Biomedicines ; 8(9)2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32942603

RESUMO

Glutathione (GSH) is an endogenous antioxidant found in plants, animals, fungi, and some microorganisms that protects cells by neutralizing hydrogen peroxide. Honokiol, an active ingredient of Magnolia officinalis, is known for antioxidant, anti-inflammatory, and anti-bacterial properties. We investigated the protective mechanism of honokiol through regulating cellular GSH in renal proximal tubules against acute kidney injury (AKI). First, we measured cellular GSH levels and correlated them with the expression of GSH biosynthetic enzymes after honokiol treatment in human kidney-2 (HK-2) cells. Second, we used pharmacological inhibitors or siRNA-mediated gene silencing approach to determine the signaling pathway induced by honokiol. Third, the protective effect of honokiol via de novo GSH biosynthesis was investigated in renal ischemia-reperfusion (IR) mice. Honokiol significantly increased cellular GSH levels by upregulating the subunits of glutamate-cysteine ligase (Gcl)-Gclc and Gclm. These increases were mediated by activation of nuclear factor erythroid 2-related factor 2, via PI3K/Akt and protein kinase C signaling. Consistently, honokiol treatment reduced the plasma creatinine, tubular cell death, neutrophil infiltration and lipid peroxidation in IR mice and the effect was correlated with upregulation of Gclc and Gclm. Conclusively, honokiol may benefit to patients with AKI by increasing antioxidant GSH via transcriptional activation of the biosynthetic enzymes.

9.
Nutrients ; 12(9)2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32933130

RESUMO

Endotoxin-induced acute liver injury is mediated by an excessive inflammatory response, hepatocellular oxidative stress, and apoptosis. Traditional medicinal plants have been used to treat various disorders. Platycodon grandifloras (PG) has been shown to be beneficial in relieving cough and asthma and to have anti-tumor, anti-inflammatory, anti-diabetic activities. The pharmacological action of PG is mainly due to saponins, flavonoids, phenolic, and other compounds. However, raw PG exhibits some side effects at high doses. Here, we extracted raw PG with varying fermentation methods and examined its anti-inflammatory effect and associated signaling kinases in Raw264.7 cells. Then, we investigated the effect of fermented black PG (FBPG) on endotoxin-induced liver injury. Mice were administered FBPG orally at 1 h before the lipopolysaccharide and D-galactosamine (LPS/GalN) injection and sacrificed after 5 h. Black PG (BPG) and FBPG showed a significant reduction in pro-inflammatory cytokines and extracellular nitric oxide (NO); p-38 and ERK signaling was involved in reducing inducible NO synthase in Raw264.7 cells. Consistently, FBPG attenuates LPS/GalN-induced liver injury; plasma ALT and AST, hepatic necrosis, pro-inflammatory cytokines, apoptosis, and lipid peroxidation were all reduced. In conclusion, PG extracts, particularly FBPG, play anti-inflammatory, antioxidant, and anti-apoptotic roles, alleviating endotoxin-induced acute liver injury. Processing raw PG into FBPG extract may be clinically useful by improving the pharmacologically active ingredients and reducing the required dosage.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Fígado/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/uso terapêutico , Platycodon , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocinas/metabolismo , Endotoxinas , Fermentação , Galactosamina , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/farmacologia , Células RAW 264.7 , Transdução de Sinais
10.
Mol Metab ; 42: 101089, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32987187

RESUMO

OBJECTIVE: Diabetic nephropathy (DN) is one of the most common complications of diabetes and a critical risk factor for developing end-stage renal disease. Activation of purinergic receptors, including P2Y2R has been associated with the pathogenesis of renal diseases, such as polycystic kidney and glomerulonephritis. However, the role of P2Y2R and its precise mechanisms in DN remain unknown. We hypothesised that P2Y2R deficiency may play a protective role in DN by modulating the autophagy signalling pathway. METHODS: We used a mouse model of DN by combining a treatment of high-fat diet and streptozotocin after unilateral nephrectomy in wild-type or P2Y2R knockout mice. We measured renal functional parameter in plasma, examined renal histology, and analysed expression of autophagy regulatory proteins. RESULTS: Hyperglycaemia and ATP release were induced in wild type-DN mice and positively correlated with renal dysfunction. Conversely, P2Y2R knockout markedly attenuates albuminuria, podocyte loss, development of glomerulopathy, renal tubular injury, apoptosis and interstitial fibrosis induced by DN. These protective effects were associated with inhibition of AKT-mediated FOXO3a (forkhead box O3a) phosphorylation and induction of FOXO3a-induced autophagy gene transcription. Furthermore, inhibitory phosphorylation of ULK-1 was decreased, and the downstream Beclin-1 autophagy signalling was activated in P2Y2R deficiency. Increased SIRT-1 (sirtuin-1) and FOXO3a expression in P2Y2R deficiency also enhanced autophagy response, thereby ameliorating renal dysfunction in DN. CONCLUSIONS: P2Y2R contributes to the pathogenesis of DN by impairing autophagy and serves as a therapeutic target for treating DN.


Assuntos
Autofagia/fisiologia , Nefropatias Diabéticas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animais , Apoptose , Autofagia/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Proteína Forkhead Box O3/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Podócitos/patologia , Receptores Purinérgicos P2Y2/genética , Transdução de Sinais , Estreptozocina/farmacologia
11.
Exp Mol Med ; 52(6): 877-886, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32514188

RESUMO

Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that is frequently overexpressed in advanced human cancers. Recent studies have demonstrated that aside from FAK's catalytic activity in cancer cells, its cellular localization is also critical for regulating the transcription of chemokines that promote a favorable tumor microenvironment (TME) by suppressing destructive host immunity. In addition to the protumor roles of FAK in cancer cells, FAK activity within cells of the TME may also support tumor growth and metastasis through various mechanisms, including increased angiogenesis and vascular permeability and effects related to fibrosis in the stroma. Small molecule FAK inhibitors have demonstrated efficacy in alleviating tumor growth and metastasis, and some are currently in clinical development phases. However, several preclinical trials have shown increased benefits from dual therapies using FAK inhibitors in combination with other chemotherapies or with immune cell activators. This review will discuss the role of nuclear FAK as a driver for tumor cell survival as well as potential therapeutic strategies to target FAK in both tumors and the TME.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Microambiente Tumoral/fisiologia , Animais , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Microambiente Tumoral/genética
12.
Int J Mol Sci ; 21(10)2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32455571

RESUMO

In various vascular diseases, extracellular matrix (ECM) and integrin expression are frequently altered, leading to focal adhesion kinase (FAK) or proline-rich tyrosine kinase 2 (Pyk2) activation. In addition to the major roles of FAK and Pyk2 in regulating adhesion dynamics via integrins, recent studies have shown a new role for nuclear FAK in gene regulation in various vascular cells. In particular, FAK primarily localizes within the nuclei of vascular smooth muscle cells (VSMCs) of healthy arteries. However, vessel injury increased FAK localization back to adhesions and elevated FAK activity, leading to VSMC hyperplasia. The study suggested that abnormal FAK or Pyk2 activation in vascular cells may cause pathology in vascular diseases. Here we will review several studies of FAK and Pyk2 associated with integrin signaling in vascular diseases including restenosis, atherosclerosis, heart failure, pulmonary arterial hypertension, aneurysm, and thrombosis. Despite the importance of FAK family kinases in vascular diseases, comprehensive reviews are scarce. Therefore, we summarized animal models involving FAK family kinases in vascular diseases.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Doenças Vasculares/metabolismo , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Doenças Vasculares/patologia
13.
Oxid Med Cell Longev ; 2019: 1754593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31285782

RESUMO

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring low concentrations of ROS in nonphagocytic cells. We found that dissolving luminol in NaOH, rather than DMSO, increased the H2O2-induced CL signal and that the addition of 4-iodophenylboronic acid (4IPBA) further increased CL intensity. Our new system also increased the rate and intensity of the CL signal in phorbol 12-myristate 13-acetate- (PMA-) treated HT-29 colon cancer cells compared to those in luminol only. We were able to quantify ROS levels from both cells and media in parallel using an H2O2 standard. A significant benefit to our system is that we can easily measure stimulus-induced ROS formation in a real-time manner and also investigate intracellular signaling pathways from a single sample simultaneously. We found that PMA induced tyrosine phosphorylation of protein tyrosine kinases (PTKs), such as focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), and Src, and increased actin stress fiber formation in a ROS-dependent manner. Interestingly, treatment with either N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) reduced the PMA-stimulated phosphorylation of these PTKs, implicating a potential role in cellular ROS signaling. Thus, our newly optimized CL assay using 4IPBA and a chemiluminescent imaging method provides a simple, real-time, and low-cost method for the quantification of low levels of ROS.


Assuntos
Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Compostos de Boro/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Células HT29 , Humanos , Immunoblotting , Iodobenzenos/farmacologia , Oniocompostos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
Sci Rep ; 9(1): 7617, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110200

RESUMO

Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1ß (IL-1ß) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1ß stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1ß signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE-/- mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.


Assuntos
Quinase 1 de Adesão Focal/genética , Quinase 2 de Adesão Focal/genética , Expressão Gênica/genética , Inflamação/genética , Interleucina-1beta/genética , Fator de Necrose Tumoral alfa/genética , Animais , Adesão Celular/genética , Moléculas de Adesão Celular/genética , Células Cultivadas , Citocinas/genética , Células Endoteliais/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Molécula 1 de Adesão de Célula Vascular/genética
15.
Circ Res ; 125(2): 152-166, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31096851

RESUMO

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.


Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fator de Transcrição GATA4/metabolismo , Miócitos de Músculo Liso/metabolismo , Túnica Íntima/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Ciclina D1/genética , Quinase 1 de Adesão Focal/antagonistas & inibidores , Hiperplasia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Túnica Íntima/patologia
16.
Biochem Biophys Res Commun ; 509(4): 1034-1040, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660359

RESUMO

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1). As VCAM-1 is a major receptor for α4 integrin and plays a key role in leukocyte recruitment, we reasoned that inhibition of FAK activity may reduce VCAM-1 expression within LNs and thus reduce metastasis of α4 integrin-expressing melanoma to LNs. First, we found that a pharmacological FAK inhibitor, PF-271, blocked tumor necrosis factor-α (TNF-α)-mediated VCAM-1 expression on human dermal lymphatic endothelial cells (HDLECs). In vitro, PF-271 significantly decreased B16F10 melanoma adhesion to and transmigration through HDLECs compared to TNF-α treated cells. Furthermore, in vivo FAK inhibition by oral PF-271 administration reduced VCAM-1 expression in inguinal, cervical, and popliteal LNs compared to vehicle treated mice. Finally, in a footpad metastasis model, B16F10 melanoma cells were injected into the right footpad of C57BL/6 mice, and PF-271 (50 mg/kg, twice daily for 6 days) was orally administrated after 1 week of tumor transplantation. While untreated mice exhibited significant metastatic melanoma lesions in popliteal LNs, PF-271 treated mice showed only marginal melanoma metastasis. These results support the possibility that FAK inhibitors may be a novel preventative option in melanoma metastasis by blocking VCAM-1 expression in LNs.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Integrina alfa4/metabolismo , Linfonodos/patologia , Melanoma/patologia , Metástase Neoplásica/prevenção & controle , Molécula 1 de Adesão de Célula Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Melanoma/química , Melanoma Experimental , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Molécula 1 de Adesão de Célula Vascular/metabolismo
17.
J Med Food ; 22(1): 14-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30207814

RESUMO

Perilla oil has been shown to be beneficial for ameliorating metabolic disorders, but its protective effect is still controversial. We investigated the effect of perilla oil on obesity-induced hepatic and vascular changes in high-fat diet (HFD)-fed mice and provided underlying mechanisms for potential therapeutic applications. Tomato and paprika extract was added to prevent the oxidation during storage of perilla oil. HFD-fed mice were orally administered palm or perilla oil for 90 days. Food intake, body and liver weight, and serum cholesterol levels were measured. Arterial and hepatic lipid accumulation was determined by histological staining. Hepatic triglyceride levels and the expression of proteins regulating lipid metabolism were analyzed. Food intake and body weight were not different between palm oil-treated and perilla oil-treated mice. Serum cholesterol level was significantly lower in perilla oil-treated mice compared with palm oil-treated mice. HFD-induced lipid accumulation was also lower in thoracic aorta and liver by perilla oil compared with palm oil. Perilla oil also decreased hepatic triglyceride level without changing the liver weight. Perilla oil treatment increased the AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation and the lipolytic protein levels, whereas it decreased the lipogenic protein levels in the liver. In conclusion, perilla oil reduced serum cholesterol and arterial and hepatic lipid accumulation in HFD-fed mice. The data suggest that perilla oil improves the balance of lipogenic and lipolytic protein expression, and ameliorates obesity-induced metabolic disorders and cardiovascular diseases.


Assuntos
Aorta/efeitos dos fármacos , Dieta Hiperlipídica , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Obesidade/complicações , Perilla/química , Ácido alfa-Linoleico/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Aorta/metabolismo , Colesterol/sangue , Gorduras na Dieta/sangue , Fígado Gorduroso/sangue , Fígado Gorduroso/prevenção & controle , Lipogênese/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Fosforilação , Fitoterapia , Extratos Vegetais/farmacologia , Óleos Vegetais/farmacologia , Triglicerídeos/sangue
18.
Nutrients ; 10(11)2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30405076

RESUMO

Diabetic nephropathy (DN) is a diabetic complication marked by albuminuria and a decline of the glomerular filtration rate. Diabetic kidneys are defective in the autophagy process and mitochondrial function and their enhancement of activity alleviates the pathology. In this paper, we developed a mouse model of DN by a combined treatment of a high-fat diet and streptozotocin after unilateral nephrectomy and supplementation with flower or leaf extracts of Abelmoschus manihot (AM) were tested. The preventive effects of the extracts on DN pathology and changes on autophagy and mitochondrial proteins were investigated. DN mice showed a significant increase in fasting blood glucose, plasma creatinine, blood urea nitrogen, and urinary albumin levels. Periodic acid⁻Schiff and Sirius red staining of the diabetic kidney presented a significant change in glomerular and tubular structures that was associated with podocyte loss and fibrotic protein accumulation. These changes were attenuated by AM extract treatment in DN mice. In addition, hepatic injury, proinflammatory cytokines, and lipid accumulation were decreased by AM extracts in DN mice. As a protective mechanism, AM extracts significantly increased the expression of proteins by regulating autophagy and mitochondrial dynamics, which potentially prevented the kidney and liver from accumulating pathogenic proteins and dysfunctional mitochondria, which alleviated the progression of DN.


Assuntos
Abelmoschus/química , Autofagia/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Fígado Gorduroso/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Glicemia/metabolismo , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Dieta Hiperlipídica , Taxa de Filtração Glomerular , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Folhas de Planta/química , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
19.
Biochim Biophys Acta Mol Basis Dis ; 1864(6 Pt A): 2169-2182, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29604334

RESUMO

Insulin resistance, defined as attenuated sensitivity responding to insulin, impairs insulin action. Direct causes and molecular mechanisms of insulin resistance have thus far remained elusive. Here we show that alternative translation initiation (ATI) of Caveolin-2 (Cav-2) regulates insulin sensitivity. Cav-2ß isoform yielded by ATI desensitizes insulin receptor (IR) via dephosphorylation by protein-tyrosine phosphatase 1B (PTP1B), and subsequent endocytosis and lysosomal degradation of IR, causing insulin resistance. Blockage of Cav-2 ATI protects against insulin resistance by preventing Cav-2ß-PTP1B-directed IR desensitization, thereby normalizing insulin sensitivity and glucose uptake. Our findings show that Cav-2ß is a negative regulator of IR signaling, and identify a mechanism causing insulin resistance through control of insulin sensitivity via Cav-2 ATI.


Assuntos
Antígenos CD/metabolismo , Caveolina 2/metabolismo , Resistência à Insulina/genética , Iniciação Traducional da Cadeia Peptídica/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Animais , Antígenos CD/genética , Caveolina 2/genética , Códon de Iniciação/genética , Endocitose , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteólise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/genética
20.
Exp Mol Med ; 50(4): 1-17, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29674665

RESUMO

Activation of the α7 nicotinic acetylcholine receptor (α7nAChR) has been shown to attenuate excessive inflammation by inhibiting proinflammatory cytokines during ischemia-reperfusion (IR) injury; however, the underlying kidney-specific molecular mechanisms remain unclear. The protective action of α7nAChR against renal IR injury was investigated using a selective α7nAChR agonist and antagonist. α7nAChR activation reduced plasma creatinine levels and tubular cell damage, whereas α7nAChR inhibition aggravated the IR-induced phenotype. α7nAChR activation decreased neutrophil infiltration and proinflammatory cytokine expression, increased heme oxygenase-1 (HO-1) expression, and reduced proximal tubular apoptosis after IR as shown by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and caspase-3 cleavage. In this study, we first showed that α7nAChR activation in the proximal tubules induced HO-1 expression through the phosphoinositide 3-kinase (PI3K)/Akt and protein kinase C (PKC) signaling pathway in vivo in renal IR mice and in vitro in proximal tubular cells. Chemical inhibitors of PKC or PI3K/Akt and small interfering RNA-mediated PKC silencing confirmed the signal specificity of α7nAChR-mediated HO-1 induction in the proximal tubular cells. α7nAChR activation inhibited high-mobility group box 1 release by inducing HO-1 expression and reduced proinflammatory cytokine gene expression and apoptotic cell death in tumor necrosis factor α-stimulated proximal tubular cells. Taken together, we conclude that α7nAChR activation in proximal tubular cells directly protects cells against renal IR injury by inducing HO-1 expression through PI3K/Akt and PKC signaling.


Assuntos
Injúria Renal Aguda , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/biossíntese , Isquemia , Túbulos Renais Proximais , Proteínas de Membrana/biossíntese , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Animais , Isquemia/metabolismo , Isquemia/patologia , Isquemia/prevenção & controle , Túbulos Renais Proximais/irrigação sanguínea , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos
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