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1.
Nat Commun ; 11(1): 4765, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958780

RESUMO

Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.


Assuntos
Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Ganho de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
2.
PLoS Pathog ; 16(8): e1008823, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845931

RESUMO

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.


Assuntos
Vírus da Influenza A/metabolismo , Pulmão , Macrófagos , Infecções por Orthomyxoviridae , Proteínas PrPC/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais Murinos/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Mutantes , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas PrPC/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
3.
Zhongguo Zhen Jiu ; 40(7): 765-70, 2020 Jul 12.
Artigo em Chinês | MEDLINE | ID: mdl-32648402

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria. METHODS: A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg-1•d-1) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells. RESULTS: In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (P<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (P<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (P<0.01). CONCLUSION: Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.


Assuntos
Tecido Conjuntivo/metabolismo , Eletroacupuntura , Mastócitos/metabolismo , Quinase Syk/metabolismo , Urticária/terapia , Quinases da Família src/metabolismo , Pontos de Acupuntura , Animais , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
4.
Nat Commun ; 11(1): 3416, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651372

RESUMO

The recent demonstration that primary cells from the liver can be expanded in vitro as organoids holds enormous promise for regenerative medicine and disease modelling. The use of three-dimensional (3D) cultures based on ill-defined and potentially immunogenic matrices, however, hampers the translation of liver organoid technology into real-life applications. We here use chemically defined hydrogels for the efficient derivation of both mouse and human hepatic organoids. Organoid growth is found to be highly stiffness-sensitive, a mechanism independent of acto-myosin contractility and requiring instead activation of the Src family of kinases (SFKs) and yes-associated protein 1 (YAP). Aberrant matrix stiffness, on the other hand, results in compromised proliferative capacity. Finally, we demonstrate the establishment of biopsy-derived human liver organoids without the use of animal components at any step of the process. Our approach thus opens up exciting perspectives for the establishment of protocols for liver organoid-based regenerative medicine.


Assuntos
Fígado/citologia , Organoides/citologia , Humanos , Hidrogéis , Fígado/metabolismo , Organoides/metabolismo , Engenharia Tecidual/métodos , Fatores de Transcrição/metabolismo , Quinases da Família src/metabolismo
5.
PLoS One ; 15(7): e0235850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673341

RESUMO

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.


Assuntos
Autorrenovação Celular , Glucose/metabolismo , Células-Tronco Neoplásicas/metabolismo , Quinases da Família src/metabolismo , Aldeído Desidrogenase 1/genética , Aldeído Desidrogenase 1/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/fisiologia , Proteoma/genética , Proteoma/metabolismo , Quinases da Família src/genética
6.
PLoS Genet ; 16(6): e1008715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559233

RESUMO

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.


Assuntos
Proteínas de Drosophila/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas ras/metabolismo , Animais , Células Cultivadas , Sequência Conservada , Drosophila , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Janus Quinase 2/metabolismo , Fosforilação , Tirosina/química , Tirosina/genética , Ubiquitinação , Proteínas ras/química , Proteínas ras/genética , Quinases da Família src/metabolismo
7.
PLoS One ; 15(6): e0235116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569321

RESUMO

Here, we examine known GTPase regulators of vesicle trafficking events to assess whether they affect endothelial cell (EC) lumen and tube formation. We identify novel roles for the small GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA, RalB and caveolin-1 in co-regulating membrane trafficking events that control EC lumen and tube formation. siRNA suppression of individual GTPases such as Rab3A, Rab8A, and RalB markedly inhibit tubulogenesis, while greater blockade is observed with combinations of siRNAs such as Rab3A and Rab3B, Rab8A and Rab11A, and RalA and RalB. These combinations of siRNAs also disrupt very early events in lumen formation including the formation of intracellular vacuoles. In contrast, knockdown of the endocytosis regulator, Rab5A, fails to inhibit EC tube formation. Confocal microscopy and real-time videos reveal that caveolin-1 strongly labels intracellular vacuoles and localizes to the EC apical surface as they fuse to form the luminal membrane. In contrast, Cdc42 and Rab11A localize to a perinuclear, subapical region where intracellular vacuoles accumulate and fuse during lumen formation. Our new data demonstrates that EC tubulogenesis is coordinated by a series of small GTPases to control polarized membrane trafficking events to generate, deliver, and fuse caveolin-1-labeled vacuoles to create the apical membrane surface.


Assuntos
Caveolina 1/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Exocitose , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(25): 14139-14149, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32503917

RESUMO

Agonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gαs, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gαs coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed.


Assuntos
Receptores de Dopamina D1/metabolismo , Transdução de Sinais , Arrestina/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Domínios Proteicos , Receptores de Dopamina D1/química , Quinases da Família src/metabolismo
9.
Vascul Pharmacol ; 131: 106762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585188

RESUMO

4-hydroxynonenal (4HNE) is a reactive aldehyde, which is involved in oxidative stress associated pathogenesis. The cellular toxicity of 4HNE is mitigated by aldehyde dehydrogenase (ALDH) 2. Thus, we hypothesize that ALDH2 inhibition exacerbates 4HNE-induced decrease in coronary endothelial cell (EC) migration in vitro. To test our hypothesis, we pharmacologically inhibited ALDH2 in cultured mouse coronary ECs (MCECs) by disulfiram (DSF) (2.5 µM) before challenging the cells with different doses of 4HNE (25, 50 and 75 µM) for 4, 12, 16 and 24 h. We evaluated MCEC migration by scratch wound migration assay. 4HNE attenuated MCEC migration significantly relative to control (P < .05), which was exacerbated with DSF pretreatment (P < .05). DSF pretreatment exacerbated 4HNE-induced decrease in ALDH2 activity in MCECs. Next, we showed that 75 µM 4HNE significantly decreased the intracellular mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), focal adhesion kinase (FAK) and other promigratory genes compared to control, which were further decreased by DSF pretreatment. 75 µM 4HNE also decreased the protein levels of VEGFR2, FAK, phospho-FAK, Src and paxillin in MCECs. Thus, we conclude that ALDH2 inhibition potentiates 4HNE-induced decrease in MCECs migration in vitro.


Assuntos
Aldeído-Desidrogenase Mitocondrial/antagonistas & inibidores , Aldeídos/farmacologia , Movimento Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Linhagem Celular , Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Camundongos , Paxilina/metabolismo , Fosforilação , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/metabolismo
10.
PLoS One ; 15(4): e0231739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352989

RESUMO

OBJECTIVES: We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvß3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation. METHODS AND RESULTS: Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition. CONCLUSION: The FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.


Assuntos
Permeabilidade Capilar/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Hemorragia/patologia , Microvasos/patologia , Quinases da Família src/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Fibrinogênio/toxicidade , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Hemorragia/induzido quimicamente , Humanos , Microscopia Intravital , Pulmão/irrigação sanguínea , Masculino , Mesentério/irrigação sanguínea , Mesentério/diagnóstico por imagem , Microvasos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genética
11.
Nat Commun ; 11(1): 1994, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332736

RESUMO

Gas1 and Boc/Cdon act as co-receptors in the vertebrate Hedgehog signalling pathway, but the nature of their interaction with the primary Ptch1/2 receptors remains unclear. Here we demonstrate, using primordial germ cell migration in mouse as a developmental model, that specific hetero-complexes of Ptch2/Gas1 and Ptch1/Boc mediate the process of Smo de-repression with different kinetics, through distinct modes of Hedgehog ligand reception. Moreover, Ptch2-mediated Hedgehog signalling induces the phosphorylation of Creb and Src proteins in parallel to Gli induction, identifying a previously unknown Ptch2-specific signal pathway. We propose that although Ptch1 and Ptch2 functionally overlap in the sequestration of Smo, the spatiotemporal expression of Boc and Gas1 may determine the outcome of Hedgehog signalling through compartmentalisation and modulation of Smo-downstream signalling. Our study identifies the existence of a divergent Hedgehog signal pathway mediated by Ptch2 and provides a mechanism for differential interpretation of Hedgehog signalling in the germ cell niche.


Assuntos
Quimiotaxia/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Receptor Patched-2/metabolismo , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Embrião de Mamíferos , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Inativação de Genes , Imunoglobulina G/metabolismo , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptor Patched-1/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Receptor Smoothened/metabolismo , Imagem com Lapso de Tempo , Quinases da Família src/metabolismo
12.
Sci China Life Sci ; 63(5): 697-705, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32246402

RESUMO

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation. Transcriptional co-activator Yes-associated protein (YAP) functions as a major downstream effector and key node of the Hippo pathway. Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions. ß-adrenergic receptor (ß-AR), a typical G protein coupled receptor (GPCR), mediates proliferation in various cell types and regulates multiple physical and pathological processes. However, the role of ß-AR in regulating YAP remains elusive. Here, we report that ß-AR can obviously stimulate YAP tyrosine phosphorylation. The mechanism is that ß-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357. Further studies demonstrate that inhibition of Src kinase activity can obviously alleviate ß-AR induced YAP tyrosine phosphorylation and cell proliferation. We conclude that ß-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.


Assuntos
Receptores Adrenérgicos beta/metabolismo , Fatores de Transcrição/metabolismo , Quinases da Família src/metabolismo , Animais , Proliferação de Células , Fibroblastos/citologia , Regulação da Expressão Gênica , Células HEK293 , Coração , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fosforilação , Ratos , Transfecção , Tirosina/metabolismo
13.
PLoS Genet ; 16(3): e1008694, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196488

RESUMO

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members.


Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Quinases da Família src/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Endocitose/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Domínios Proteicos , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
14.
Toxicology ; 437: 152440, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32197950

RESUMO

Arsenic is an endocrine disruptor that promotes breast cancer (BCa) development. Estrogen synthesis, through aromatase activation, is essential for BCa promotion and progression through activating the G-coupled estrogen receptor 1 (GPER1), regulating rapid nongenomic effects involved in cell proliferation and migration of BCa cells. Herein, was studied the role of aromatase activation and the GPER1 pathway on sodium arsenite-induced promotion and progression of MDA-MB-231 and MDA-MB-453 BCa cell lines. Our results demonstrated that 0.1 µM of sodium arsenite induces cell proliferation, migration, invasion, and stimulates aromatase activity of BCa cell lines MDA-MB-231, MDA-MB-453, MCF-7, but not in a nontumorigenic breast epithelial cell line (MCF-12A). Using letrozole (an aromatase inhibitor) and G-15 (a GPER1-selective antagonist), we demonstrated that sodium arsenite-induced proliferation and migration is mediated by induction of aromatase enzyme and, at least in part, by GPER1 activation in MDA-MB-231 and MDA-MB-453 cells. Sodium arsenite induced phosphorylation of Src that participated in sodium arsenite-induced aromatase activity, and -cell proliferation of MDA-MB-231 cell line. Overall, data suggests that sodium arsenite induces a positive-feedback loop, resulting in the promotion and progression of BCa cells, through induction of aromatase activity, E2 production, GPER1 stimulation, and Src activation.


Assuntos
Aromatase/metabolismo , Arsenitos/toxicidade , Neoplasias da Mama/enzimologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativadores de Enzimas/toxicidade , Compostos de Sódio/toxicidade , Neoplasias da Mama/patologia , Ativação Enzimática , Estradiol/metabolismo , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica , Fosforilação , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo
15.
Anticancer Res ; 40(3): 1335-1344, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132030

RESUMO

BACKGROUND/AIM: The prognosis of pancreatic cancer has not improved due to its migratory feature and refractory potential to chemo-resistance with absence of effective diagnosis. Despite continuous efforts, its underlying mechanisms of malignant nature remain ambiguous. The objective of this study was to investigate delta-like 1 (DLL1) as a tumor suppressor in the metastasic ability of human pancreatic cancer cells. MATERIALS AND METHODS: Cellular expression of DLL1 was demonstrated using the GEO public database and western blot analysis. The biological function of DLL1 was validated by biological behavior analysis. Prognosis to DLL1 expression was demonstrated using analysis of the GEO public database. RESULTS: Analysis using the GEO database and western blotting showed higher DLL1 mRNA and protein expression levels in pancreatic cancer compared to those in normal pancreas. DLL1 was uniquely expressed in seven human pancreatic cancer cell lines compared to human pancreatic duct epithelial H6c7 cells. Ablation of DLL1 expression stimulated migration and invasion by activating Src and p38 phosphorylation, but not viability and chemo-resistance of human pancreatic cancer cells. In addition, expression of DLL1 was correlated with migratory features of pancreatic cancer in vivo. Moreover, high DLL1 expression was associated with a favorable prognosis in pancreatic cancer patients. CONCLUSION: DLL1 is a potent suppressor of pancreatic cancer metastasis. Understanding correlation between expression and function of DLL1 might contribute to our knowledge of the complicated mechanism of pancreatic cancer metastasis.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , Transdução de Sinais , Taxa de Sobrevida
16.
Biochem Biophys Res Commun ; 523(4): 972-978, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31973815

RESUMO

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.


Assuntos
NADPH Oxidases/metabolismo , Fosfotirosina/metabolismo , Superóxidos/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células A549 , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Fosforilação , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família src/metabolismo
17.
FASEB J ; 34(1): 66-81, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914639

RESUMO

Angiogenesis is critical for the development, progression, and metastasis of hepatocellular carcinoma (HCC), but the roles of miR-3064-5p in HCC angiogenesis are still unknown. In this study, the roles of miR-3064-5p in HCC angiogenesis were studied in 192 HCC patients, xenograft mouse models, and HCC cell lines. The results showed that miR-3064-5p expression was significantly decreased in HCC tissues and cells, and downregulated miR-3064-5p was associated with upregulated angiogenic potential of HCC. MiR-3064-5p inhibited proangiogenic VEGFA and angiogenin expressions but induced antiangiogenic endostatin and MMP12 expressions, finally leading to suppression of HCC angiogenesis, as shown by the decline in intratumoral microvessel density (MVD). Moreover, miR-3064-5p was inversely correlated with lncRNA MALAT1 and FOXA1. FOXA1 bound to and interacted with CD24 and then regulated Src phosphorylation. MiR-3064-5p played an antiangiogenic role by inhibiting the FOXA1/CD24/Src pathway, whereas oncogenic MALAT1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-3064-5p to alleviate the suppressive effect on the FOXA1 pathway. HCC patients with high miR-3064-5p, low MALAT1, or low FOXA1 expression had a better prognosis with longer overall survival and recurrence-free survival. In univariate and multivariate analyses, miR-3064-5p was identified as the independent prognostic predicator for HCC progression and patient survival. Taken together, miR-3064-5p exerts an antiangiogenic role by targeting the FOXA1/CD24/Src pathway but oncogenic lncRNA MALAT1 acts as a ceRNA to sponge miR-3064-5p. MiR-3064-5p is of great clinical significance and is a novel prognostic indicator and an attractive therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/irrigação sanguínea , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Antígeno CD24/genética , Antígeno CD24/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Neoplasias Hepáticas/patologia , Camundongos , MicroRNAs/genética , Neoplasias Experimentais , RNA Longo não Codificante/genética , Quinases da Família src/genética , Quinases da Família src/metabolismo
18.
Cancer Res ; 80(5): 1156-1170, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932454

RESUMO

The noncanonical Wnt ligand Wnt5a is found in high concentrations in ascites of women with ovarian cancer. In this study, we elucidated the role of Wnt5a in ovarian cancer metastasis. Wnt5a promoted ovarian tumor cell adhesion to peritoneal mesothelial cells as well as migration and invasion, leading to colonization of peritoneal explants. Host components of the ovarian tumor microenvironment, notably peritoneal mesothelial cells and visceral adipose, secreted Wnt5a. Conditional knockout of host WNT5A significantly reduced peritoneal metastatic tumor burden. Tumors formed in WNT5A knockout mice had elevated cytotoxic T cells, increased M1 macrophages, and decreased M2 macrophages, indicating that host Wnt5a promotes an immunosuppressive microenvironment. The Src family kinase Fgr was identified as a downstream effector of Wnt5a. These results highlight a previously unreported role for host-expressed Wnt5a in ovarian cancer metastasis and suggest Fgr as a novel target for inhibition of ovarian cancer metastatic progression.Significance: This study establishes host-derived Wnt5a, expressed by peritoneal mesothelial cells and adipocytes, as a primary regulator of ovarian cancer intraperitoneal metastatic dissemination and identifies Fgr kinase as novel target for inhibition of metastasis.


Assuntos
Carcinoma Epitelial do Ovário/patologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/imunologia , Peritônio/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteína Wnt-5a/metabolismo , Quinases da Família src/metabolismo , Animais , Carcinoma Epitelial do Ovário/imunologia , Adesão Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Peritoneais/secundário , Peritônio/citologia , Peritônio/imunologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Chem Commun (Camb) ; 56(10): 1521-1524, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922153

RESUMO

Tremendous advancements in proteolysis targeting chimera (PROTAC) technology have been made in recent years. However, whether a covalent inhibitor-based PROTAC can be developed remains controversial. Here, we successfully developed chimeric degraders based on covalent inhibitors to degrade BTK and BLK kinases, demonstrating that covalent inhibitor-based PROTACs are viable and useful tools.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Técnicas Biossensoriais/métodos , Inibidores de Proteínas Quinases/química , Quinases da Família src/metabolismo , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Humanos , Células K562 , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Quinases da Família src/antagonistas & inibidores
20.
Am J Respir Cell Mol Biol ; 62(1): 61-73, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264901

RESUMO

Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. We tested the hypothesis that Src-epidermal growth factor receptor (EGFR) signaling mediates enhanced vasoconstrictor sensitivity after chronic hypoxia through NADPH oxidase-derived superoxide generation. Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone after chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the EGFR inhibitor gefitinib. Inhibition of NADPH oxidase, Rac1 (Ras-related C3 botulinum toxin substrate 1), and EGFR abolished pressure-induced pulmonary arterial tone and endothelin-1 (ET-1)-dependent calcium sensitization and vasoconstriction after chronic hypoxia. Consistently, chronic hypoxia augmented ET-1-induced superoxide production through EGFR signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were also activated by ET-1 after chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction in response to ET-1. A role for matrix metalloproteinase 2 to mediate Src-dependent EGFR activation is further supported by our findings. Our studies support a novel role for an Src kinase-EGFR-NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension after chronic hypoxia.


Assuntos
Receptores ErbB/metabolismo , Hipóxia/tratamento farmacológico , Pulmão/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacocinética , Quinases da Família src/metabolismo , Animais , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Pulmão/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley
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