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1.
Life Sci ; 239: 117010, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31672578

RESUMO

AIMS: Amlexanox, an inhibitor of nuclear factor κB kinase epsilon (IKKε) and TANK-binding kinase 1(TBK1), was demonstrated to be effective in diabetes and obesity. The aim of this study was to explore the molecular mechanisms of its role in non-alcoholic fatty liver disease (NAFLD). MAIN METHODS: NAFLD mouse models were established by using eight-week-old male C57BL/6 mice fed with high-fat diet (HFD) or (and) lipopolysaccharide (LPS) for 18 weeks. From the beginning of HFD, HFD-induced mice were subjected to amlexanox or vehicle for 18 weeks. HFD + LPS-induced mice were treated with amlexanox or vehicle for the last 6 weeks. Blood biochemistry parameters were determined using automatic biochemistry analyzer. Histological changes of liver tissue were observed by hematoxylin-eosin (H&E) staining and Oil Red O staining. The expressions of IKKε and smooth muscle actin-α (α-SMA) were evaluated through immunohistochemistry. Serum inflammatory mediator was determined by enzyme linked immunosorbent assay (ELISA). Gene expressions involved in glucose and lipid metabolism, insulin signaling pathway were examined using quantitative RT-PCR or Western blotting. KEY FINDINGS: This study demonstrated that amlexanox reversed glucose and lipid metabolic disturbance and hepatic steatosis in NAFLD mice model. IKKε was specific expressed in hepatic stellate cells (HSCs) instead of hepatocytes. This study also found that amlexanox improved insulin signaling (Insulin-IRS-1-Akt) in hepatocytes through inhibiting inflammation (IKKε-NF-κB-TNF-α/IL-1α) in HSCs. SIGNIFICANCE: The present study confirmed that IKKε was specific expressed in HSCs. Inhibition of activated HSCs was responsible for effects of amlexanox on NAFLD, with improving insulin signal pathway in hepatocytes.


Assuntos
Aminopiridinas/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Quinase I-kappa B/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Actinas/antagonistas & inibidores , Aminopiridinas/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/efeitos dos fármacos
2.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
3.
Ren Fail ; 41(1): 419-426, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31140898

RESUMO

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) patients who progress to end-stage renal disease (ESRD). With the increasing incidence of CKD, it is of importance to develop effective therapies that blunt development of renal fibrosis. FFNT25 is a newly developed molecular compound that could be used to prevent fibrosis. In this study, we administered FFNT25 to rats following unilateral ureteral obstruction (UUO) to investigate its anti-fibrosis mechanism. Thirty-two Sprague-Dawley rats were randomly divided into four groups: (1) control (normal rats), (2) sham-operated, (3) UUO-operated + vehicle, and (4) UUO-operated + FFNT25. Two weeks after UUO, the rats were gavaged with either FFNT25 (20.6 mg/kg/day) or vehicle for two weeks. Serum, urine, and kidney samples were collected at the end of the study. FFNT25 reduced levels of renal fibrosis and decreased mRNA and protein levels of extracellular matrix (ECM) markers α-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1) following UUO compared to vehicle treatment (n = 8, p<.05). The current results indicate that FFNT25 can affect both the production and degradation of collagen fibers to reduce fibrosis.


Assuntos
Rim/patologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Inibidores de Serino Proteinase/farmacologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fibrose , Humanos , Rim/efeitos dos fármacos , Masculino , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/patologia , Inibidores de Serino Proteinase/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Obstrução Ureteral/complicações
4.
Molecules ; 24(10)2019 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-31109130

RESUMO

Norcantharidin (NCTD), a demethylated derivative of cantharidin, has been reported to exhibit activity against various types of cancers. However, the anti-invasive effects of NCTD and its molecular mechanism in human mucoepidermoid carcinoma (MEC) remain incompletely elucidated. Clonogenic, wound healing, invasion, zymography, western blotting and immunocytochemistry assays were performed in YD-15 cells to investigate the anti-invasive effect of NCTD and its molecular mechanism of action. The inhibitory effects of NCTD on invasiveness were compared with those of a novel focal adhesion kinase (FAK) kinase inhibitor, PF-562271. NCTD markedly suppressed the colony formation, migration, and invasion of YD-15 cells as well as the activities of MMP-2 and MMP-9. It disrupted F-actin reorganization through suppressing the FAK/Paxillin axis. Moreover, NCTD exhibited a powerful anti-invasive effect compared with that of PF-562271 in YD-15 cells. Collectively, these results suggest that NCTD has a potential anti-invasive activity against YD-15 cells. This study may clarify the impact of NCTD on migration and invasion of human MEC cells.


Assuntos
Actinas/antagonistas & inibidores , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Movimento Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Paxilina/antagonistas & inibidores , Carcinoma Mucoepidermoide/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Transdução de Sinais
5.
Biochem Biophys Res Commun ; 509(4): 973-977, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30654940

RESUMO

Angiotensin II (Ang II) modulates VSMCs phenotypic switch that play a critical role in the cardiovascular diseases. MicroRNA-27a (miR-27a) has been proven to be involved in regulating vascular remodeling; however, the functional role of miR-27a in VSMCs in response to Ang II stimulation need to be elucidated. Cell proliferation and migration were measured by Cell counting kit-8 (CCK-8), BrdU incorporation and scratch wound assay in VSMCs transfected with miR-27a or its inhibitor. The target of miR-27a was confirmed using bioinformatics analysis and luciferase reporter assay. Ang II treatment time-dependently increased proliferation and migration of VSMCs accompanied with downregulation of α-smooth muscle-actin (α-SMA) and upregulation of miR-27a expression. Moreover, knockdown of miR-27a in VSMCs significantly attenuated Ang II-induced cell proliferation and migration, whereas this effect was aggravated by overexpression of miR-27a. A potential mechanistic analysis revealed that miR-27a directly targeted α-SMA, which mediated miR-27a-induced cell proliferation and migration. In conclusion, these results indicate that miR-27a acts as a novel regulator of Ang II-induced proliferation and migration by directly targeting α-SMA expression in VSMCs in vitro, and may be a potential therapeutic target for treating vascular diseases.


Assuntos
Actinas/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MicroRNAs/fisiologia , Músculo Liso Vascular/citologia , Angiotensina II/farmacologia , Humanos , Miócitos de Músculo Liso/citologia
6.
mBio ; 9(5)2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30352935

RESUMO

Influenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that a step in IAV particle assembly is blocked in MDM. Using an in situ proximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.IMPORTANCE Identification of host cell determinants promoting or suppressing replication of viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM, which are not permissive to IAV replication, fail to support virus particle formation. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.


Assuntos
Membrana Celular/metabolismo , Vírus da Influenza A/fisiologia , Macrófagos/virologia , Montagem de Vírus , Actinas/antagonistas & inibidores , Linhagem Celular , Membrana Celular/virologia , Células Cultivadas , Citocalasina D/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/ultraestrutura , Macrófagos/citologia , Microscopia Eletrônica de Varredura , Proteínas da Matriz Viral/metabolismo , Vírion , Liberação de Vírus/efeitos dos fármacos , Replicação Viral
7.
Sci Rep ; 8(1): 15585, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30348987

RESUMO

The oxygen consumption rate (OCR) and cytochrome c oxidase (CcO) activity of respiratory complex IV (CIV) in brain mitochondria significantly decline in middle-aged male mice compared to younger male mice. To explore the mechanisms underlying the regulation of brain mitochondrial function, we examined CIV-associated proteins, and identified actin inside the isolated brain mitochondria. Inhibiting actin polymerization using cytochalasin B (CB) significantly enhanced the OCR and CcO activity of CIV in the mitochondria. These changes were accompanied by a significant reduction in the amount of CIV-bound cytochrome c (cyt c). Actin was also associated with respiratory complex III (CIII); however, the amount of CIII-bound cyt c increased significantly after treatment of the mitochondria with CB. In contrast, no significant alteration in the assembly or the CcO activity of CIV in CIV-containing supercomplexes or CIV monomers was induced by CB. These results suggest that mitochondrial actin plays a crucial role in the regulation of the CcO activity and OCR of CIV with modification of the retention of cyt c between CIV and CIII.


Assuntos
Actinas/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Actinas/antagonistas & inibidores , Animais , Encéfalo/crescimento & desenvolvimento , Citocalasina B/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio , Polimerização
8.
Plasmid ; 98: 37-44, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-30196057

RESUMO

The CRISPR/Cas9 system is a powerful genome editing tool for disrupting the expression of specific genes in a variety of cells. However, the genome editing procedure using currently available vectors is laborious, and there is room for improvement to obtain knockout cells more efficiently. Therefore, we constructed a novel vector for high efficiency genome editing, named pGedit, which contains EGFP-Bsr as a selection marker, expression units of Cas9, and sgRNA without a terminator sequence of the U6 promoter. EGFP-Bsr is a fusion protein of EGFP and blasticidin S deaminase, and enables rapid selection and monitoring of transformants, as well as confirmation that the vector has not been integrated into the genome. By using pGedit, we targeted human ACTB, ACTG1 and mouse Nes genes coding for ß-actin, γ-actin and nestin, respectively. Knockout cell lines of each gene were easily and efficiently obtained in all three cases. In this report, we show that our novel vector, pGedit, significantly facilitates genome editing.


Assuntos
Actinas/antagonistas & inibidores , Sistemas CRISPR-Cas , Edição de Genes/métodos , Vetores Genéticos , Nestina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Actinas/genética , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Sequência de Bases , Marcação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Nestina/genética , Regiões Promotoras Genéticas , Homologia de Sequência
9.
Exp Cell Res ; 371(2): 426-434, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30201453

RESUMO

Although parallel alignment of fibroblasts to the tension lines of scar has been evidenced in vivo, how scar contracture generates directional contraction remains largely unclear due to the lack of effective in vitro model. Fibroblast populated collagen lattice (FPCL), a widely used in vitro model, fails to mimic scar contracture since it produces concentric contraction with the random orientation of fibroblast. We hypothesized that a novel FPCL model with fibroblast alignment might produce directional contraction and then simulate scar contracture better. Here, we showed that although direct current electric fields (DCEFs) enabled fibroblasts aligned perpendicularly to the field vector, it also promoted electrotactic migration of fibroblast in FPCL. By contrast, biphasic pulse direct current electric fields (BPDCEFs), featured by reversal of the EF direction periodically, abolished the electrotactic migration, but induced fibroblast alignment in a pulse frequency dependent manner. Specifically, BPDCEF at a pulse frequency of 0.0002 Hz induced fibroblast alignment comparable to that induced by DCEF under the same field strength (300 mV/mm), leading to an enhanced contraction of FPCL along the direction of cell alignment. FPCL pretreated by BPDCEF showed an elliptical contraction whereas it was concentric in control FPCL. Further study revealed that F-actin redistributions acted as a key mechanism for the induction of fibroblasts alignment by BPDCEF. Cytochalasin D, an inhibitor of actin dynamics, abolished F-actins redistribution, and significantly suppressed the fibroblasts alignment and the directional contraction of FPCL. Importantly, BPDCEF significantly increased RhoA activity in fibroblasts, while this response was attenuated by C3 transferase pre-treatment, a potent inhibitor of RhoA, caused F-actin depolymerization and actin filament bundle randomly distributed. Taken together, our study suggests a crucial role for fibroblast orientation in scar contracture, and provides a novel FPCL model that may be feasible and effective for investigating scar contracture in vitro.


Assuntos
Eletricidade , Fibroblastos/citologia , Modelos Biológicos , Tecidos Suporte , ADP Ribose Transferases/farmacologia , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Toxinas Botulínicas/farmacologia , Movimento Celular , Cicatriz/genética , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno/química , Citocalasina D/farmacologia , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Ratos , Pele/citologia , Pele/metabolismo , Tensão Superficial , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
10.
Drug Des Devel Ther ; 12: 2707-2713, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30214158

RESUMO

Purpose: To examine the effects and mechanism of action of fasudil on cytoskeletal polymerization, collagen synthesis, and apoptosis in fibroblasts derived from human urethral scar tissue. Materials and methods: Fibroblasts treated with or without transforming growth factor ß1 (TGF-ß1, 10 ng/mL) were incubated with fasudil (12.5, 25, 50 µmol/L) for 24 hours. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of Arp2, Arp3, WASP, and WAVE2. Collagen I and III protein levels were also evaluated by Western blotting. The filamentous actin cytoskeleton was examined by immunofluorescence and epifluorescence microscopy. An Annexin V-FITC/PI staining assay was used to investigate apoptosis. Results: TGF-ß1-dependent induction of actin polymerization and collagen synthesis and promotion of apoptosis were dose dependent. When compared with untreated controls, fasudil significantly decreased the expression of Arp2, Arp3, WASP, WAVE2, Collagen I, and Collagen III in cells treated with or without TGF-ß1. Fasudil also promoted apoptosis in cells, irrespective of TGF-ß1 treatment. Conclusion: Irrespective of TGF-ß1 activation status, fasudil suppressed actin polymerization and collagen synthesis and induced apoptosis in human urethral scar fibroblasts via the Rho/ROCK signaling pathway.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Actinas/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Colágeno/biossíntese , Polimerização/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Uretra/patologia , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Actinas/metabolismo , Adulto , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Uretra/metabolismo
11.
Development ; 145(10)2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29724756

RESUMO

During the early embryonic cell cycles, zebrafish germ plasm ribonucleoparticles (RNPs) gradually multimerize and become recruited to the forming furrows. RNPs multimerization occurs prior to and during furrow initiation, as forming aggregates move outward through their association with the tips of growing interphase astral microtubules. Germ plasm RNPs are also associated with short cortical F-actin. We show that, in embryos mutant for the cytoskeletal regulator mid1ip1l, germ plasm RNPs fail to become recruited to the furrow, accumulating instead at the periphery of the blastodisc. RNP aggregates are associated with zones of mid1ip1l-dependent cyclical local cortical F-actin network enrichments, as well as contractions at both the cortex and the contractile ring. F-actin inhibition in wild-type embryos mimics the RNP peripheral accumulation defect of mid1ip1l mutants. Our studies suggest that a common mechanism underlies distinct steps of germ plasm RNP segregation. At the cortex, this process attenuates microtubule-dependent outward RNP movement to retain RNPs in the blastodisc cortex and allow their recruitment to the furrows. F-actin network contraction likely also facilitates higher-order germ plasm RNP multimerization.


Assuntos
Actinas/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Embrião não Mamífero/embriologia , Ribonucleoproteínas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Citoesqueleto de Actina/metabolismo , Actinas/antagonistas & inibidores , Animais , Blastodisco/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/genética , Embrião não Mamífero/metabolismo , Células Germinativas/metabolismo , Microtúbulos/metabolismo , Multimerização Proteica/fisiologia , Transporte Proteico/genética , Proteínas de Peixe-Zebra/genética
12.
Cell Death Dis ; 9(5): 517, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29725063

RESUMO

Increased Actin-like 6A (ACTL6A) expression has been implicated in the development of diverse cancers and recently associated with the Hippo signaling pathway, which is known to regulate biological properties, including proliferation, tissue regeneration, stem cell biology, as well as tumorigenesis. Here we first show that ACTL6A is upregulated in human gliomas and its expression is associated with glioma patient survival. ACTL6A promotes malignant behaviors of glioma cells in vitro and in orthotopic xenograft model. In co-immunoprecipitation assays, we discover that ACTL6A physically associated with YAP/TAZ and furthermore disrupts the interaction between YAP and ß-TrCP E3 ubiquitin ligase, which promotes YAP protein degradation. Moreover, effects of ACTL6A on glioma cells proliferation, migration, and invasion could be mediated by YAP/TAZ. These data indicate that ACTL6A may contribute to cancer progression by stabilizing YAP/TAZ and therefore provide a novel therapeutic target for the treatment of human gliomas.


Assuntos
Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Encefálicas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas/genética , Proteínas Contendo Repetições de beta-Transducina/genética , Actinas/antagonistas & inibidores , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosfoproteínas/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Fatores de Transcrição , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Contendo Repetições de beta-Transducina/metabolismo
13.
J Pharm Pharm Sci ; 21(1): 119-134, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29578859

RESUMO

PURPOSE: To develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to hepatic stellate cells (HSC). VA was selected to increase the uptake by HSC based on their function in the storage of VA. METHODS: DOTAP/DOPE liposomes were prepared by film hydration method and their surfaces were decorated with VA. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 assay. Inhibition of BMP4 and α-SMA was determined by PCR and ELISA. RESULTS: VA-coated lipoplexes exhibited an average particle sizes less than 200 nm and zeta potential around +25 mV both determined using ZetaPALS. Inclusion of VA to liposomal surfaces significantly enhanced their cellular uptake without affecting cytotoxicity. VA-coupled liposomes carrying BMP4-siRNA resulted in a significant reduction in BMP4 and α-SMA at both mRNA and protein levels.  Conclusion: VA-coated liposomes were successfully designed to deliver BMP4-siRNA to specifically target HSC. The novel delivery system discussed herein may serve as a potential therapeutic strategy for the treatment of liver fibrosis in the future. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.


Assuntos
Actinas/antagonistas & inibidores , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Células Estreladas do Fígado/efeitos dos fármacos , Nanopartículas/química , Vitamina A/farmacologia , Actinas/biossíntese , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Humanos , Lipídeos/química , Lipossomos/química , RNA Interferente Pequeno/química
14.
PLoS One ; 13(2): e0192146, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29444136

RESUMO

Fibroblast growth factor 21 (FGF21) is an important metabolic regulator expressed predominantly in the liver. In this study, we evaluated the role of LY2405319, an analogue of FGF21, in hepatic stellate cell (HSC) activation and in a methionine and choline-deficient (MCD)-diet induced mouse model of liver fibrosis. During liver injury, HSCs trans-differentiate into activated myofibroblasts which produce alpha-smooth muscle actin (α-SMA) and become a major cell type in hepatic fibrogenesis. Succinate and succinate receptor (GPR91) signaling has emerged as a regulator to promote α-SMA production in MCD diet- induced mice. Treatment with palmitate or MCD medium on LX-2 cells (HSCs) increased succinate concentration in the conditioned medium and cell lysate of LX-2 cells and increased production of GPR91 and α-SMA. However, LY2405319 administration ameliorates palmitate or MCD media-induced succinate production and decreases over-expression of GPR91 and α-SMA in LX2-cells. In an in vivo study, the MCD diet treatment caused increased steatohepatitis and liver fibrosis compared with the control diet in mice. Administration of LY2405319 improved steatohepatitis ameliorated GPR91 and α -SMA production in the liver, decreased succinate concentration in both liver and serum of MCD diet -induced mice. These results suggest that FGF21 reduces production of α-SMA by inhibiting the succinate-GPR91 pathway. We conclude that FGF21 acts as an inhibitor of the succinate-GPR91 pathway to control liver fibrosis. This suggests that FGF21 has therapeutic potential for treating liver fibrogenesis.


Assuntos
Actinas/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Succinatos/antagonistas & inibidores , Actinas/biossíntese , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I , Cirrose Hepática/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
J Vet Med Sci ; 80(2): 225-234, 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29279465

RESUMO

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.


Assuntos
Actinas/antagonistas & inibidores , Furanos/farmacologia , Piranos/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Microscopia Eletrônica de Transmissão , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Leves de Miosina/efeitos dos fármacos , Fenilefrina/farmacologia , Polimerização/efeitos dos fármacos , Ratos , Ratos Wistar
16.
J Biol Chem ; 293(7): 2606-2616, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29282288

RESUMO

Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.


Assuntos
Actinas/metabolismo , Profilinas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/antagonistas & inibidores , Actinas/genética , Animais , Aorta Torácica/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Polimerização/efeitos dos fármacos , Profilinas/antagonistas & inibidores , Profilinas/química , Profilinas/genética , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
17.
Mol Vis ; 24: 789-800, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30636861

RESUMO

Purpose: This study aimed to investigate the effect of nintedanib on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts and reveal the molecular mechanisms involved. Methods: Primary cultured HTFs were incubated with transforming growth factor ß1 (TGF-ß1) alone or combined with nintedanib, and cell proliferation and migration were measured by cell counting kit-8 (CCK8) and the scratch wound assay, respectively. HTF contractility was evaluated with a 3D collagen contraction assay. The mRNA and protein levels of α smooth muscle actin (α-SMA) and Snail and the phosphorylation levels of Smad2/3, p38 mitogen-activated protein kinase (p38MAPK), and extracellular signal-regulated kinase ½ (ERK1/2) were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunofluorescence staining. Results: Nintedanib inhibited the proliferation and migration of HTFs in a dose-dependent manner. Furthermore, nintedanib prevented HTF myofibroblast differentiation via downregulation of mRNA and protein expression of α-SMA and Snail. A three-dimensional (3D) collagen gel contraction assay demonstrated that nintedanib effectively inhibits myofibroblast contraction induced by TGF-ß1. Mechanistically, we revealed that nintedanib reduces the TGF-ß1-induced phosphorylation of Smad2/3, p38MAPK, and ERK1/2, suggesting that nintedanib acts through both classic and nonclassic signaling pathways of TGF-ß1 to prevent HTF activation. Conclusions: Our study provides new evidence that nintedanib has potent antifibrotic effects in HTFs and suggests that it may be used as a potential therapeutic agent for subconjunctival fibrosis.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Miofibroblastos/efeitos dos fármacos , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Cápsula de Tenon/citologia , Cápsula de Tenon/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Bioorg Med Chem ; 25(24): 6322-6331, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29042221

RESUMO

The antitumor and actin-depolymerizing marine macrolide aplyronine A (ApA) synergistically binds to tubulin in association with actin, and prevents spindle formation and mitosis. While the crystal structure of the actin ApA complex was solved in 2006, its interaction with the tubulin heterodimer has not been clarified. To investigate the binding modes of ApA as a unique protein-protein interaction (PPI)-inducer between these two cytoskeletal proteins, we prepared its photoaffinity acetylene and fluorescent derivatives with the aid of molecular modeling studies for probe design. Among these three derivatives, the ApA-PPA-TAMRA probe specifically photoreacted with both actin and tubulin in vitro. However, the photolabeling yield of tubulin was quite low (up to ∼1%), and one of the major side-reactions was the addition of a water molecule to the carbene species generated from an aryldiazirine moiety on the hydrophilic surface of actin.


Assuntos
Actinas/antagonistas & inibidores , Antineoplásicos/farmacologia , Desenho de Drogas , Macrolídeos/farmacologia , Marcadores de Fotoafinidade/farmacologia , Tubulina (Proteína)/metabolismo , Actinas/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Macrolídeos/síntese química , Macrolídeos/química , Modelos Moleculares , Estrutura Molecular , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
19.
Invest Ophthalmol Vis Sci ; 58(12): 5298-5307, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29049733

RESUMO

Purpose: The actin cytoskeleton of trabecular meshwork (TM) cells plays a role in regulating aqueous humor outflow. Many studies have investigated stress fibers, but F-actin also assembles into other supramolecular structures including filopodia. Recently, specialized filopodia called tunneling nanotubes (TNTs) have been described, which communicate molecular signals and organelles directly between cells. Here, we investigate TNT formation by TM cells. Methods: Human TM cells were labeled separately with the fluorescent dyes, DiO and DiD, or with mitochondrial dye. Fixed or live TM cells were imaged using confocal microscopy. Image analysis software was used to track fluorescent vesicles and count the number and length of filopodia. The number of fluorescently labeled vesicles transferred between cells was counted in response to specific inhibitors of the actin cytoskeleton. Human TM tissue was stained with phalloidin. Results: Live-cell confocal imaging of cultured TM cells showed transfer of fluorescently labeled vesicles and mitochondria via TNTs. In TM tissue, a long (160 µm) actin-rich cell process bridged an intertrabecular space and did not adhere to the substratum. Treatment of TM cells with CK-666, an Arp2/3 inhibitor, significantly decreased the number and length of filopodia, decreased transfer of fluorescently labeled vesicles and induced thick stress fibers compared to vehicle control. Conversely, inhibiting stress fibers using Y27632 increased transfer of vesicles and induced long cell processes. Conclusions: Identification of TNTs provides a means by which TM cells can directly communicate with each other over long distances. This may be particularly important to overcome limitations of diffusion-based signaling in the aqueous humor fluid environment.


Assuntos
Comunicação Celular/fisiologia , Nanotubos , Pseudópodes/fisiologia , Transdução de Sinais/fisiologia , Malha Trabecular/citologia , Vesículas Transportadoras/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/antagonistas & inibidores , Actinas/metabolismo , Adolescente , Adulto , Amidas/farmacologia , Criança , Pré-Escolar , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Humanos , Indóis/farmacologia , Microscopia Confocal , Pessoa de Meia-Idade , Piridinas/farmacologia , Coloração e Rotulagem , Malha Trabecular/fisiologia
20.
J Cell Mol Med ; 21(12): 3506-3514, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28767184

RESUMO

Acute kidney disease (AKI) leads to increased risk of progression to chronic kidney disease (CKD). Antithrombin III (ATIII) is a potent anticoagulant with anti-inflammatory properties, and we previously reported that insufficiencies of ATIII exacerbated renal ischaemia-reperfusion injury (IRI) in rats. In this study, we examined the characteristic of AKI-CKD transition in rats with two distinct AKI models. Based on our observation, left IRI plus right nephrectomy (NX-IRI) was used to determine whether ATIII had therapeutic effects in preventing CKD progression after AKI. It was observed that NX-IRI resulted in significant functional and histological damage at 5 weeks after NX-IRI compared with sham rats, which was mitigated by ATIII administration. Besides, we noticed that ATIII administration significantly reduced NX-IRI-induced interstitial fibrosis. Consistently, renal expression of collagen-1, α-smooth muscle actin and fibronectin were substantial diminished in ATIII-administered rats compared with un-treated NX-IRI rats. Furthermore, the beneficial effects of ATIII were accompanied with decreased M1-like macrophage recruitment and down-regulation of M1-like macrophage-dependent pro-inflammatory cytokines such as tumour necrosis factor α, inducible nitric oxide synthase and interleukin-1ß, indicating that ATIII prevented AKI-CKD transition via inhibiting inflammation. Overall, ATIII shows potential as a therapeutic strategy for the prevention of CKD progression after AKI.


Assuntos
Lesão Renal Aguda/prevenção & controle , Antitrombina III/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Animais , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fibronectinas/antagonistas & inibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Rim/metabolismo , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Nefrectomia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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