Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 425
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
2.
Acta Biochim Biophys Sin (Shanghai) ; 51(4): 356-364, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30877754

RESUMO

Metastasis is the main cause of death in patients with colorectal cancer (CRC), but the molecular mechanism is not yet fully understood. Previous studies have shown that P zero-related protein (PZR), a member of the immunoglobulin family, can promote fibronectin-dependent migration of mouse embryonic fibroblasts as well as invasion and metastasis of hepatic carcinoma cells. However, the role of PZR in CRC remains unclear. In this study, we determined the ectopic expression of PZR in CRC tissues, and results showed that PZR expression was increased not only in tumors with higher pathological stage, but also in tumors with distant metastasis. Through PZR-knockdown and overexpression in CRC cell lines, we found that the expression of PZR had significant effect on the invasion and migration of CRC cells as well as the phosphorylation of pro-metastasis proteins including focal adhesion kinase (FAK) and Src. Taken together, this study indicates that PZR may promote the invasion and migration of CRC cells through increasing the phosphorylation of FAK and Src, which provides a new theoretical basis and a possible marker for the diagnosis or prognosis of CRC metastasis.


Assuntos
Neoplasias Colorretais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Quinases da Família src/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Células HCT116 , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metástase Neoplásica , Fosfoproteínas/genética , Fosforilação , Prognóstico , Interferência de RNA , Quinases da Família src/genética
3.
Proc Natl Acad Sci U S A ; 116(9): 3518-3523, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808745

RESUMO

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.


Assuntos
Neoplasias da Mama/genética , Galactosiltransferases/genética , Glicoesfingolipídeos/genética , Microdomínios da Membrana/genética , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caveolina 1/genética , Caveolina 1/metabolismo , Progressão da Doença , Proteína de Domínio de Morte Associada a Fas/genética , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicoesfingolipídeos/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/genética , Saporinas/genética , Transdução de Sinais/genética , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Embrionários Estágio-Específicos/metabolismo
4.
Proc Natl Acad Sci U S A ; 116(9): 3524-3529, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733287

RESUMO

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Integrinas/química , Proteínas de Membrana/química , Conformação Proteica , Proteínas de Ligação a Telômeros/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/genética , Membrana Celular/química , Cristalografia por Raios X , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , Fosforilação , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética
5.
Cell Physiol Biochem ; 49(6): 2277-2292, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30257244

RESUMO

BACKGROUND/AIMS: In this study, we aimed to investigate the effects of genistein on the focal adhesion signaling pathway through its regulation of FAK. Genistein ultimately restored and alleviated estradiol-induced vascular endothelial injury. METHODS: Microarray analysis was used to select differentially expressed genes. MTT assay was performed to detect the cell activity, and ROS test and NO test were performed to detect the degree of damage to HUVECs (human umbilical vein endothelial cells). The relative mRNA expression levels and protein expression levels of FAK were tested by western blot and qRT-PCR. GO functional analysis and KEGG pathway analysis were applied to predict the possible relationship between functions and related pathways, and transwell assay was used to detect cell invasion and migration. RESULTS: FAK was highly expressed in the HUVECs treated with estradiol (HU-ESTs). Cell viability and NO level decreased, whereas ROS level increased in the HU-ESTs. Effective knockdown of FAK in HU-ESTs elevated cell viability and NO levels while suppressing ROS levels. In addition, inhibition of FAK greatly decreased cell invasion and migration, while the overexpression of FAK enhanced cell invasion and migration. KEGG further indicated focal adhesion pathways were activated. Genistein elevated HU-EST viability, and NO and ROS level increased in a concentration dependent manner. Transwell and western blot assays revealed that genistein could reduce the FAK expression levels and alleviate the damage to the HU-ESTs. CONCLUSION: FAK overexpression promoted invasion and migration of the HU-ESTs. However, genistein greatly suppressed FAK and estradiol-induced vascular endothelial cell injury.


Assuntos
Adesão Celular/efeitos dos fármacos , Estradiol/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Genisteína/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína Substrato Associada a Crk/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Óxido Nítrico/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zixina/metabolismo
6.
J Agric Food Chem ; 66(26): 6708-6716, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29877083

RESUMO

Tricin, a natural flavonoid present in large amounts in rice bran, was investigated for the mechanisms by which it exhibited antiproliferation and anti-invasion in C6 glioma cells. The results indicated that treatment with 5, 10, 25, and 50 µM tricin for 48 h significantly ( p < 0.05) inhibited cell numbers and colony numbers with values of 134.3 ± 5.5, 114.6 ± 2.5, 106.3 ± 3.2, and 57.3 ± 10.2, respectively. Tricin also inhibited C6-cell motility, migration, and invasion. Tricin changed the cytoskeletal organization, reduced matrix-metalloproteinase (MMP) expression, and upregulated E-cadherin. Tricin decreased FAK protein levels and suppressed focal-adhesion-kinase (FAK)-downstream-signal activation. Most importantly, tricin dose-dependently upregulated microRNA-7 (miR-7). Transfection with an miR-7 inhibitor suppressed miR-7 expression, increased FAK expression, and promoted the proliferation and invasion in C6 cells. The data support a novel anticancer mechanism of tricin that involves upregulation of FAK-targeting miR-7 in C6 glioma cells.


Assuntos
Antineoplásicos/farmacologia , Flavonoides/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Glioma/fisiopatologia , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/enzimologia , Glioma/genética , Humanos , MicroRNAs/genética , Invasividade Neoplásica
7.
Dev Cell ; 45(5): 565-579.e3, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29870718

RESUMO

Contact inhibition of locomotion is defined as the behavior of cells to cease migrating in their former direction after colliding with another cell. It has been implicated in multiple developmental processes and its absence has been linked to cancer invasion. Cellular forces are thought to govern this process; however, the exact role of traction through cell-matrix adhesions and tension through cell-cell adhesions during contact inhibition of locomotion remains unknown. Here we use neural crest cells to address this and show that cell-matrix adhesions are rapidly disassembled at the contact between two cells upon collision. This disassembly is dependent upon the formation of N-cadherin-based cell-cell adhesions and driven by Src and FAK activity. We demonstrate that the loss of cell-matrix adhesions near the contact leads to a buildup of tension across the cell-cell contact, a step that is essential to drive cell-cell separation after collision.


Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Embrião não Mamífero/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Crista Neural/fisiologia , Xenopus laevis/fisiologia , Quinases da Família src/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Células Cultivadas , Embrião não Mamífero/citologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Crista Neural/citologia , Fosforilação , Xenopus laevis/embriologia , Quinases da Família src/genética
8.
Acta Biochim Biophys Sin (Shanghai) ; 50(5): 465-472, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29648579

RESUMO

Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of clear cell renal cell carcinoma (ccRCC). This study aimed to further elucidate the molecular mechanisms underlying Capn4-mediated ccRCC progression. The mRNA expression levels in ccRCC cells were measured by quantitative real-time PCR. The effects of Capn4 on cell adhesion, invasion, and migration were examined by cell adhesion assay, cell invasion assay, and wound-healing assay, respectively. The protein levels were detected by western blot analysis. The effect of Capn4 on cancer metastasis in vivo was assessed in a nude mice xenograft model. It was found that Capn4 was up-regulated in the ccRCC cells, and Capn4 overexpression suppressed cell adhesion activity and increased cell invasion and migration in 786-O cells, while Capn4 silencing increased cell adhesion activity and impaired the invasion and migration ability of Caki-1 cells. Capn4 overexpression also increased the protein level of cleaved talin in 786-O cells, while Capn4 silencing decreased the protein level of cleaved talin in Caki-1 cells. The focal adhesion kinase (FAK)/AKT/MAPK signaling was activated by Capn4 overexpression in 786-O cells, and was inhibited by Capn4 down-regulation in Caki-1 cells. Capn4 overexpression increased the protein levels of matrix metalloproteinase 2 (MMP-2), vimentin, N-cadherin, and down-regulated E-cadherin in 786-O cells, while Capn4 silencing decreased the protein levels of MMP-2, vimentin, N-cadherin, and up-regulated E-cadherin in Caki-1 cells. Capn4 also promoted cancer metastasis in the in vivo nude mice xenograft model. Our results implicate the functional role of Capn4 in ccRCC invasion and migration, which may contribute to cancer metastasis in ccRCC.


Assuntos
Calpaína/genética , Carcinoma de Células Renais/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Neoplasias Renais/genética , Transdução de Sinais/genética , Talina/genética , Animais , Calpaína/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Talina/metabolismo , Transplante Heterólogo
9.
J Agric Food Chem ; 66(15): 3860-3869, 2018 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-29606008

RESUMO

Atherosclerosis is characterized by the buildup of plaque inside arteries. Our recent studies demonstrated that polyphenolic natural products can reduce oxidative stress, inflammation, angiogenesis, hyperlipidemia, and hyperglycemia. A previous study also showed that mulberry water extract (MWE) can inhibit atherosclerosis and contains considerable amounts of polyphenols. Therefore, in the present study, we investigated whether mulberry polyphenol extract (MPE) containing high levels of polyphenolic compounds could affect vascular smooth muscle cell (VSMC; A7r5 cell) motility. We found that MPE inhibited expression of FAK, Src, PI3K, Akt, c-Raf, and suppressed FAK/Src/PI3K interaction. Further investigations showed that MPE reduced expression of small GTPases (RhoA, Cdc42, and Rac1) to affect F-actin cytoskeleton rearrangement, down-regulated expression of MMP2 and vascular endothelial growth factor (VEGF) mRNA through NFκB signaling, and thereby inhibited A7r5 cell migration. Taken together, these findings highlight MPE inhibited migration in VSMC through FAK/Src/PI3K signaling pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Morus/química , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Fosfatidilinositol 3-Quinases/genética , Extratos Vegetais/isolamento & purificação , Polifenóis/isolamento & purificação , Ratos , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/genética
10.
Endocrinology ; 159(2): 696-709, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29186427

RESUMO

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Resistência à Insulina , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Movimento Celular , Células Endoteliais/citologia , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Integrinas/genética , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
11.
Expert Opin Ther Pat ; 28(2): 139-145, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29210300

RESUMO

INTRODUCTION: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes at sites of cell adhesion to the extracellular matrix (ECM) and mediates signalling events downstream of integrin engagement of the ECM. FAK is known to regulate cell survival, proliferation and migration. Areas covered: FAK expression has also been shown to be up-regulated in many cancer types. Previous study also indicates that FAK-mediated signaling and functions are intrinsically involved in the progression of tumor aggressiveness, suggesting that FAK is a promising target for anticancer therapies. Small molecule FAK inhibitors have been developed and are being tested in clinical phase trials. Expert Opinion: These inhibitors have demonstrated to be effective by inducing tumor cell apoptosis in addition to reducing metastasis and angiogenesis. In this review, we give updates on the design, synthesis and structure-activity relationship analysis of small molecule FAK inhibitors discovered from 2015 until now. We also review the FAK inhibitors that are in clinical development and highlight the future prospects.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Progressão da Doença , Desenho de Drogas , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Alvo Molecular , Neoplasias/enzimologia , Patentes como Assunto , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
12.
J Mol Neurosci ; 64(1): 102-110, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29209901

RESUMO

The process of neuronal migration is precisely regulated by different molecules during corticogenesis. The FAK (focal adhesion kinase) plays a critical role in embryogenesis and is involved in cell motility through focal adhesions, but the underlying mechanisms on inordinate expression are unclear. To investigate the effect of FAK overexpression on neuronal migration spatiotemporally, mice FAK was transfected into the neurons in vivo by electroporation. Results showed that exogenous FAK distributed in the cytoplasm (in vivo) and co-localized with vinculin (in vitro) and induced aberrant neuronal migration via phosphorylation of FAK at Tyr925 during cerebral cortex development. Meanwhile, FAK Y925F mutant also induced aberrant neuronal migration like inordinate FAK/GFP phenotype. All these results implied that FAK-induced abnormal phenotype depended on phosphorylation of FAK at Tyr925, and this demonstrated that the overexpression of FAK impaired neuronal migration through its phosphorylation and activity of FAK during corticogenesis.


Assuntos
Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Cricetinae , Cricetulus , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neurônios/fisiologia , Fosforilação , Vinculina/metabolismo
13.
Cell Physiol Biochem ; 44(4): 1509-1525, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29197863

RESUMO

BACKGROUND/AIMS: Periodic mechanical stress could significantly promote chondrocyte proliferation and matrix synthesis. However, the mechanisms underlying the ability of chondrocyte detecting and responding to periodic mechanical stimuli have not been well delineated. METHODS: Quantitative proteomic analysis was performed to construct the differently expressed proteome profiles of chondrocyte under pressure. Then a combination of Western blot, quantitative real-time PCR, lentiviral vector and histological methods were used to confirm the proteomic results and investigate the mechanoseing mechanism. RESULTS: Growth factor receptor-bound protein 2 (Grb2), a component of integrin adhesome, was found a 1.49-fold increase in dynamic stress group. This process was mediated through integrin ß1, leading to increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) respectively and then produce the corresponding biological effects. CONCLUSION: This was the first time to demonstrate Grb2 has such an important role in periodic mechanotransduction, and the proteomic data could facilitate the further investigation of chondrocytes mechanosensing.


Assuntos
Proteína Adaptadora GRB2/metabolismo , Proteômica , Estresse Mecânico , Agrecanas/genética , Agrecanas/metabolismo , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Cromatografia Líquida de Alta Pressão , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína Adaptadora GRB2/antagonistas & inibidores , Proteína Adaptadora GRB2/genética , Imuno-Histoquímica , 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 , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização por Electrospray , Engenharia Tecidual , Regulação para Cima
14.
Neoplasia ; 19(12): 961-971, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29069627

RESUMO

Src, a non-receptor tyrosine kinase protein, plays a critical role in cell proliferation and tumorigenesis. SUMOylation, a reversible ubiquitination-like post-translational modification, is vital for tumor progression. Here, we report that the Src protein can be SUMOylated at lysine 318 both in vitro and in vivo. Hypoxia can induce a decrease of Src SUMOylation along with an increase of Y419 phosphorylation, a phosphorylation event required for Src activation. On the other hand, treatment with hydrogen peroxide can enhance Src SUMOylation. Significantly, ectopic expression of SUMO-defective mutation, Src K318R, promotes tumor growth more potently than that of wild-type Src, as determined by migration assay, soft agar assay, and tumor xenograft experiments. Consistently, Src SUMOylation leads to a decrease of Y925 phosphorylation of focal adhesion kinase (FAK), an established regulatory event of cell migration. Our results suggest that SUMOylation of Src at lysine 318 negatively modulate its oncogenic function by, at least partially, inhibiting Src-FAK complex activity.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Progressão da Doença , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Lisina/metabolismo , Camundongos , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Sumoilação , Quinases da Família src/química , Quinases da Família src/genética
15.
J Pharmacol Exp Ther ; 363(3): 428-443, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29021381

RESUMO

Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS-mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS-mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin (P < 0.05) levels but significantly increased matrix metallopeptidase 9 (MMP9) levels (P < 0.01) in A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS-mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteína-Tirosina Quinases de Adesão Focal/biossíntese , Proteína-Tirosina Quinases de Adesão Focal/genética , Genes src/genética , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Células A549 , Animais , Dasatinibe/farmacologia , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Masculino , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Nus , Niacinamida/farmacologia , Proteína Oncogênica v-akt/biossíntese , Proteína Oncogênica v-akt/genética , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 18(9)2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28880199

RESUMO

Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Queratinócitos/metabolismo , Animais , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Adesões Focais/fisiologia , Humanos , Mecanotransdução Celular/fisiologia , Camundongos Knockout , Transdução de Sinais/fisiologia
17.
J Physiol Pharmacol ; 68(3): 375-383, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28820394

RESUMO

Increased extracellular pressure or shear stress activate a complex signal pathway that stimulates integrin binding affinity and potentiates metastatic cell adhesion. Inhibiting either focal adhesion kinase (FAK) and Akt1 can block this pathway, but risks interfering with the diverse other functions of each kinase. However, the mechanotransduced signal pathway involves a novel Akt1-FAK interaction not required for most FAK or Akt1 function, so modeling and blocking this interaction seems a desirable target. Building upon previous work suggesting that FAK-Akt1 binding is mediated by the FAK F1 lobe, we demonstrated that independently expressing the F1 domain in human Caco-2 or murine CT-26 colon cancer cells by transient or stable inducible plasmid expression respectively prevents the stimulation of cancer cell adhesion by increased extracellular pressure. Serial further truncation of the FAK F1 lobe identified shorter regions capable of pulling down Akt1 on a glutathione S-transferase (GST) - conjugated column. Ultimately, we identified a 33 residue segment (residues 94-126) at the C-terminal of the F1 lobe as sufficient to pull down Akt1. These findings raise the possibility of developing a treatment modality around the disruption of the FAK-Akt1 interaction using peptides modeled from FAK.


Assuntos
Adesão Celular/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/genética , Glutationa Transferase/metabolismo , Humanos , Camundongos , Modelos Moleculares , Pressão , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/química
18.
Methods Mol Biol ; 1652: 37-42, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28791632

RESUMO

Genetic analysis of Egfr signaling in Drosophila has a long-standing track record of making important contributions to our understanding of the Egfr pathway. While the central Ras/MAPK pathway has long been well defined, there is much to learn with regard to its cross talk with other pathways and how it is regulated. A better understanding of the regulation of Egfr signaling is of particular interest with regard to the participation of misregulated Egfr signaling in tumorigenesis. Recent studies in the fly have led to some surprising results, identifying regulators of Egfr acting in unexpected ways.


Assuntos
Drosophila/metabolismo , Receptores ErbB/metabolismo , Transdução de Sinais , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores ErbB/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/genética , Proteínas ras/genética , Proteínas ras/metabolismo
19.
Mol Med Rep ; 16(4): 4008-4014, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28765922

RESUMO

Electric field (EF) exposure can affect the elongation, migration, orientation, and division of cells. The present study tested the hypothesis that EF may also affect epithelial­mesenchymal transition (EMT) in lens epithelial cells and that this effect may be an important inducer in the pathological process of posterior capsule opacification (PCO). Human lens epithelial (HLE)­B3 cells were exposed to an EF. Experiments were performed in the presence or absence of an anti­integrin ß1 blocking antibody or a small molecule inhibitor targeting focal adhesion kinase (FAK). Cell morphology changes were observed by microscopy. The expression levels of integrin ß1, FAK, phosphorylated (p)FAK and of EMT markers, E­cadherin and Vimentin, were examined by immunofluorescence, reverse transcription­quantitative polymerase chain reaction and western blotting. Following exposure to EF, HLE­B3 cells appeared elongated and resembled more fibroblast­like cells. Expression of E­cadherin was decreased, while expression of Vimentin was increased in HLE­B3 cells exposed to EF, compared with control cells. In addition, the mRNA expression levels of integrin ß1 were increased, and the protein expression levels of integrin ß1 and pFAK were increased in HLE­B3 cells exposed to EF, compared with control cells. Blocking of integrin ß1 suppressed the EMT­related morphological changes of HLE­B3 cells and reduced the activation of FAK following EF exposure. However, blocking of pFAK did not affect the EMT status of HLE­B3 cells induced by EF. In conclusion, the present study demonstrated that EF exposure induced EMT in HLE­B3 cells and that this effect may partially be mediated by the activation of integrin ß1­FAK signaling. The present results may provide a new mechanistic approach to prevent the development of PCO.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos da radiação , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrina beta1/metabolismo , Cristalino/citologia , Transdução de Sinais , Biomarcadores , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Expressão Gênica , Humanos , Integrina beta1/genética , Vimentina/metabolismo
20.
Tumour Biol ; 39(6): 1010428317703941, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28653881

RESUMO

Cell adhesion-mediated drug resistance is an important factor that influences the effects of chemotherapy in multiple myeloma. DTX3L, a ubiquitin ligase, plays a key role in cell-cycle-related process. Here, we found that the expression of DTX3L gradually increased during the proliferation of myeloma cells, which resulted in arrest of the cell cycle in the G1 phase and promoted the adherence of myeloma cells to fibronectin or bone marrow stromal cells. In addition, silencing of DTX3L improved sensitivity to chemotherapy drugs in multiple myeloma cell lines adherent to bone marrow stromal cells and increased the expression of caspase-3 and poly-adenosine diphosphate-ribose polymerase, two markers of apoptosis. Finally, we also found that DTX3L expression was regulated by focal adhesion kinase. Taken together, the results of this study show that DTX3L plays an important role in the proliferation and cell adhesion-mediated drug resistance of multiple myeloma cells and as such may play a key role in the development of multiple myeloma.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Mieloma Múltiplo/tratamento farmacológico , Ubiquitina-Proteína Ligases/biossíntese , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Caspase 3/genética , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Tratamento Farmacológico , Fibronectinas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA