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1.
Nat Commun ; 11(1): 3282, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612176

RESUMO

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Histona Desacetilases/genética , Mecanotransdução Celular/genética , Osteócitos/metabolismo , Transdução de Sinais/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Perfilação da Expressão Gênica/métodos , Histona Desacetilases/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética , Fosforilação
2.
Breast Cancer Res ; 22(1): 59, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493400

RESUMO

BACKGROUND: Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer. METHODS: To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested. RESULTS: We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival. CONCLUSIONS: In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.


Assuntos
Carcinoma Basocelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Wnt1/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Modelos Animais de Doenças , Progressão da Doença , Feminino , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Proteína Wnt1/genética
3.
Am J Obstet Gynecol ; 223(5): 733.e1-733.e14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32417359

RESUMO

BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leiomioma/genética , Mecanotransdução Celular/efeitos dos fármacos , Sinvastatina/farmacologia , Neoplasias Uterinas/genética , Proteínas de Ancoragem à Quinase A/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/efeitos dos fármacos , Integrina beta1/genética , Integrina beta1/metabolismo , Leiomioma/metabolismo , Mecanotransdução Celular/genética , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Quinase de Cadeia Leve de Miosina/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Neoplasias Uterinas/metabolismo , Quinases Associadas a rho/efeitos dos fármacos , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
4.
ACS Appl Mater Interfaces ; 12(13): 14924-14932, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32155329

RESUMO

We have evaluated the response to nanotopography of CHO-K1 cells that express wild-type paxillin or paxillin with mutations at serine 273 that inhibit phosphorylation. Cells were grown on nanoporous and polished titanium surfaces. With all cell types, immunofluorescence showed that adhesion and spreading were minimally affected on the treated surface and that the actin filaments were more abundant and well-aligned. Scanning electron microscopy revealed changes in cell shape and abundant filopodia with lateral nanoprotrusions in response to nanoporosity. Gene expression of proteins associated with cellular adhesion and protrusions was significantly increased on the nanoporous surface regardless of the cell type. In particular, α-actinin, Rac1, Cdc42, and ITGα1 were upregulated in S273 cells with alanine substitutions, whereas FAK, Pxn, and Src were downregulated, leading to improved focal adhesion formation. These findings suggest that the surface nanoporosity can "compensate for" the genetic mutations that affect the biomechanical relationship of cells to surfaces.


Assuntos
Adesão Celular/fisiologia , Nanoporos , Paxilina/metabolismo , Animais , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Regulação para Baixo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Paxilina/genética , Fosforilação , Propriedades de Superfície , Titânio/química , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
PLoS One ; 15(2): e0228575, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049961

RESUMO

BACKGROUND: The aim was to identify a novel prognostic miRNA signature for colon cancer (CC) in silico. METHODS: Data on the expression of miRNAs and relevant clinical information for 407 patients were obtained from The Cancer Genome Atlas (TCGA), and the samples were randomly split into a validation set (n = 203) and training set (n = 204). The differential expression of miRNAs between normal tissues and patients with CC was analyzed. We detected a miRNA expression signature in the training dataset by using a Cox proportional hazard regression model. Then, we verified the signature in the validation set. Association of the miRNA signature with overall survival was assessed in the validation cohort and combined cohort by log-rank test and based on Kaplan-Meier curves. The receiver operating characteristic and disease-free survival analyses were performed to evaluate the miRNA signature of CC in the combined cohort. Multivariate and univariate Cox analyses related to survival for the miRNA signature were performed, and a nomogram was built as a prognostic model for CC. To explore the function of target genes of the miRNA signature, Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used. RESULTS: Between the matched normal tissues and colon cancer tissues, 267 differentially expressed miRNAs were detected, and a single-factor CoxPH model showed that 13 miRNAs were related to overall survival in the training cohort. Then, a five-miRNA signature was identified using a CoxPH regression model with multiple factors. The five-miRNA signature had significant prognostic value in the training cohort and was validated in the validation cohort and combined cohort. A total of 193 target genes of the miRNA signature were identified. According to the results of functional analysis of the target genes, the signaling pathways MAPK, AMPK and PI3K-Akt, focal adhesion, and microRNAs in cancer were remarkably enriched. CONCLUSION: A five-miRNA signature had increased prognostic value for CC, which may provide important biological insights for the discovery and development of molecular predictors to improve the prognosis of patients with CC.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , MicroRNAs/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Redes Reguladoras de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
6.
Med Sci Monit ; 26: e921626, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32034900

RESUMO

BACKGROUND The stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved. MATERIAL AND METHODS A compressive force of 25 g/m² was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin alpha5ß1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin alpha5ß1 inhibitor (Ac-PHSCN-NH 2, ATN-161). RESULTS Mechanical force increased the expression of integrin alpha5ß1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin alpha5ß1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited. CONCLUSIONS The integrin alpha5ß1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin alpha5ß1 pathway.


Assuntos
Citoesqueleto de Actina/metabolismo , Fibroblastos/citologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Gengiva/citologia , Integrina alfa5beta1/metabolismo , Mecanotransdução Celular , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Adolescente , Células Cultivadas , Criança , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Mecanotransdução Celular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Estresse Mecânico
7.
Cell Mol Life Sci ; 77(24): 5259-5279, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32040695

RESUMO

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Complexos Multiproteicos/genética , Neoplasias/genética , beta-Arrestinas/genética , Complexo 2 de Proteínas Adaptadoras/genética , Animais , Membrana Celular/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Receptor Tipo 1 de Angiotensina/genética , Receptores Acoplados a Proteínas-G/genética , Vasopressinas/farmacologia
8.
Int J Biol Sci ; 16(2): 342-352, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929761

RESUMO

Prostate cancer (PCa) is the third leading malignancy engendering mortality among men globally. The present study aimed at determining the expression of hepatocellular carcinoma-related protein-1 (HCRP-1) in PCa, to explore its potential role in prostate tumorigenesis in vitro and in vivo. We evaluated HCRP-1 protein with immunohistochemistry (IHC) technology and found HCRP-1 expression was significantly low in PCa tissues (PCTs); In addition, the decreased HCRP-1 was significantly associated with TNM (tumor node metastasis) stage, advanced histology grade and gleason score. Transwell, tube formation, Western blot and co-immunoprecipitation (Co-IP) assays were utilized to determine the role of down-regulating HCRP-1 in PCa cell migration, invasion and angiogenesis. Meanwhile, we found HCRP-1 depletion induced Src and focal adhesion kinase (FAK) phosphorylation, which could be reversed by Src inhibitor PP2 or FAK inhibitor. Furthermore, down-regulated HCRP-1 evidently induced lung metastasis of PCa cells in xenograft mode. Taken together, our study indicates HCRP-1 plays an important role in PCa metastasis. HCRP-1 may serve as a potential therapeutic target for PCa.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Quinases da Família src/genética , Quinases da Família src/metabolismo
9.
J Cell Physiol ; 235(3): 3096-3111, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31556111

RESUMO

Focal adhesion kinase (FAK) is critical for collagen expression but its regulation of collagen remodeling is not defined. We examined the role of FAK in the degradation and reorganization of fibrillar collagen. Compared with wild-type (WT) mouse embryonic fibroblasts, FAK null (FAK-/- ) fibroblasts generated twofold (p < .0001) higher levels of ¾ collagen I fragment and expressed up to fivefold more membrane-type matrix metalloproteinase (MMP). When plated on stiff collagen substrates, compared with WT, FAK-/- cells were smaller (threefold reduced cell surface area; p < .0001) and produced fivefold fewer cell extensions (p < .0001) that were 40% shorter (p < .001). When cultured on soft collagen gels (stiffness of ~100 Pa) for 6-48 hr, cell spreading and cell extension formation were reduced by greater than twofold (p < .05 and p < .0001, respectively) while collagen compaction and alignment were reduced by approximately 30% (p < .0001) in FAK-/- cells. Similar results were found after treatment with PF573228, a FAK inhibitor. Reconstitution of FAK-/- cells with FAK mutants showed that compared with WT, cell extension formation was reduced twofold (p < .0001) in the absence of the kinase domain and sixfold (p < .0001) with a Y397F mutant. Enhanced collagen degradation was exhibited by the mutants (~threefold increase; p < .0001 of ¾ collagen fragments without kinase domain or Y397F mutant; p < .01). Compared with FAK+/+ cells, matrices produced by FAK-/- cells generated higher levels of ß1 integrin activation (p < 0.05), extracellular-signal-regulated kinase (ERK) phosphorylation, and production of ¾ collagen I fragment by human gingival fibroblasts. Collectively these data indicate that (a) the kinase activity of FAK enhances collagen remodeling by tractional forces but inhibits collagen degradation by MMPs; (b) FAK influences the biological activity of fibroblast-secreted extracellular matrices, which in turn impacts ß1 integrin and ERK signaling, and collagen degradation.


Assuntos
Colágeno/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Metaloproteinases da Matriz/metabolismo , Animais , Adesão Celular/fisiologia , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
10.
Cells ; 8(12)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847469

RESUMO

Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
11.
Food Funct ; 10(12): 8172-8181, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31730141

RESUMO

Metastasis is the most prevalent cause of treatment failure in patients with colon cancer. Gossypol is reported to exhibit antioxidant, anticancer, antivirus and antimicrobial properties. However, the effects of gossypol on cancer invasion and tumour growth of human colon cancer remain unclear. This study aimed to provide molecular evidence associated with the antimetastatic and anti-tumour effects of gossypol on human colorectal carcinoma (CRC) cells. Gossypol inhibited the viability of human colon cancer cells in a dose-dependent manner. Gossypol was sufficient to reduce the invasion, migration and adhesion in DLD-1 and COLO 205 cells. Zymography and western blot assay showed that gossypol reduced the activities and protein expression of urokinase-type plasminogen activator (u-PA), respectively. Gossypol suppressed the level of p-focal adhesion kinase (FAK) and epithelial-to-mesenchymal transition markers, including N-cadherin, fibronectin and vimentin. Gossypol also inhibited the lung metastasis of DLD-1 cells, as indicated by the nude mouse model. These results suggested that gossypol inhibited the metastatic properties of human colon cancer cells by targeting u-PA through the FAK pathway, suggesting that gossypol could be used as an adjuvant therapeutic agent for the treatment of human colon cancer cells.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Gossipol/administração & dosagem , Neoplasias Pulmonares/prevenção & controle , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica/prevenção & controle , Ativador de Plasminogênio Tipo Uroquinase/genética , Vimentina/genética , Vimentina/metabolismo
12.
Adv Immunol ; 144: 23-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31699219

RESUMO

B cells are essential to the adaptive immune system for providing the humoral immunity against cohorts of pathogens. The presentation of antigen to the B cell receptor (BCR) leads to the initiation of B cell activation, which is a process sensitive to the stiffness features of the substrates presenting the antigens. Mechanosensing of the B cells, potentiated through BCR signaling and the adhesion molecules, efficiently regulates B cell activation, proliferation and subsequent antibody responses. Defects in sensing of the antigen-presenting substrates can lead to the activation of autoreactive B cells in autoimmune diseases. The use of high-resolution, high-speed live-cell imaging along with the sophisticated biophysical materials, has uncovered the mechanisms underlying the initiation of B cell activation within seconds of its engagement with the antigen presenting substrates. In this chapter, we reviewed studies that have contributed to uncover the molecular mechanisms of B cell mechanosensing during the initiation of B cell activation.


Assuntos
Formação de Anticorpos , Apresentação do Antígeno , Linfócitos B/imunologia , Mecanotransdução Celular/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Animais , Doenças Autoimunes/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/imunologia , Humanos , Sinapses Imunológicas/química , Sinapses Imunológicas/genética , Sinapses Imunológicas/patologia , Integrinas/imunologia , Proteínas Motores Moleculares/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo
13.
Sci Rep ; 9(1): 14669, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604999

RESUMO

GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10-1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.


Assuntos
Células Epiteliais/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Mucosa Intestinal/efeitos dos fármacos , Úlcera/tratamento farmacológico , Animais , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Células Epiteliais/patologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Humanos , Mucosa Intestinal/patologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonas/farmacologia , Úlcera/genética , Úlcera/patologia , Técnicas de Fechamento de Ferimentos , Cicatrização/efeitos dos fármacos , Cicatrização/genética
14.
FASEB J ; 33(12): 13254-13266, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31539281

RESUMO

Macrophages (Mφs) can be used as a part of cell-based cancer immunotherapy. However, they may be hampered by a failure to effectively and stably regulate their polarization state to enhance their tumoricidal effects. In this work, mechanical stretch (MS), as a biology-free modulatory method, was shown to enhance M1 polarization and tumoricidal effects. By using an in vitro Flexcell Tension system, we found that murine Mφ RAW264.7 cells showed higher M1 polarization-related mRNA expression and cytokine release after MS. Further molecular analyses found that focal adhesion kinase and NF-κB activation occurred in the MS-induced M1 polarization. Coculture of MS-preconditioned Mφ with B16F10 skin melanoma cells in vitro showed that the proliferation of B16F10 cells decreased, whereas caspase-3-induced apoptosis increased. Importantly, the injection of MS-preconditioned Mφ into murine skin melanomas in vivo impeded tumor growth; lesions were characterized by increased amounts of M1 Mφ, decreased tumor cell proliferation, and increased tumor cell apoptosis in the tumor microenvironment. Together, our results suggest that MS could be used as a simple preconditioning approach to prepare tumoricidal M1 Mφ for cancer immunotherapy.-Shan, S., Fang, B., Zhang, Y., Wang, C., Zhou, J., Niu, C., Gao, Y., Zhao, D., He, J., Wang, J., Zhang, X., Li, Q. Mechanical stretch promotes tumoricidal M1 polarization via the FAK/NF-κB signaling pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Citocinas/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética
15.
Artigo em Inglês | MEDLINE | ID: mdl-31297342

RESUMO

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.


Assuntos
Suscetibilidade a Doenças/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Actinas/metabolismo , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Suscetibilidade a Doenças/parasitologia , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases , Fosforilação , Proteínas de Protozoários/genética , Quinolonas/metabolismo , Proteínas Recombinantes/metabolismo , Sulfonas/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
16.
Aging (Albany NY) ; 11(12): 3969-3992, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31219799

RESUMO

The angiotensin II type I receptor (AGTR1) has a strong influence on tumor growth, angiogenesis, inflammation and immunity. However, the role of AGTR1 on lymph node metastasis (LNM) in breast cancer, which correlates with tumor progression and patient survival, has not been examined. AGTR1 was highly expressed in lymph node-positive tumor tissues, which was confirmed by the Oncomine database. Next, inhibition of AGTR1 reduced tumor growth and LNM in orthotopic xenografts by bioluminescence imaging (BLI). Losartan, an AGTR1-specific inhibitor, decreased the chemokine pair CXCR4/SDF-1α levels in vivo and inhibited AGTR1-induced cell migration and invasion in vitro. Finally, the molecular mechanism of AGTR1-induced cell migration and LNM was assessed by knocking down AGTR1 in normal cells or CXCR4 in AGTR1high cells. AGTR1-silenced cells treated with losartan showed lower CXCR4 expression. AGTR1 overexpression caused the upregulation of FAK/RhoA signaling molecules, while knocking down CXCR4 in AGTR1high cells downregulated these molecules. Collectively, AGTR1 promotes LNM by increasing the chemokine pair CXCR4/SDF-1α and tumor cell migration and invasion. The potential mechanism of AGTR1-mediated cell movement relies on activating the FAK/RhoA pathway. Our study indicated that inhibiting AGTR1 may be a potential therapeutic target for LNM in early-stage breast cancer.


Assuntos
Neoplasias da Mama/patologia , Quimiocina CXCL12/metabolismo , Linfonodos/patologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores CXCR4/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Movimento Celular , Quimiocina CXCL12/genética , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Losartan/administração & dosagem , Losartan/farmacologia , Metástase Linfática , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Experimentais , Receptor Tipo 1 de Angiotensina/genética , Receptores CXCR4/genética , Transdução de Sinais , Regulação para Cima , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
17.
Environ Toxicol ; 34(10): 1085-1093, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31184425

RESUMO

Geraniin has been reported to have numerous biological activities, including antiviral, antihypertensive, antihyperglycaemic, liver protective, antidiabetic, and apoptotic activities. However, the anti-migration effects of geraniin on oral cancer remain elusive. In this study, we revealed the potential antitumor mechanisms of geraniin through the inhibition of the migration and invasion of human oral cancer cell lines SCC-9 and SCC-14. The results of gelatin zymography and Western blot assays revealed that geraniin significantly reduced the activity and expression of matrix metalloproteinase-2 (MMP-2) of oral cancer cells in a concentration-dependent manner. Furthermore, geraniin potently suppressed the phosphorylation of focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK)1/2 but did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase 1/2. Moreover, blocking the MAPK/ERK1/2 pathway significantly enhanced the anti-migration ability of geraniin in oral cancer cells. In conclusion, we demonstrated that geraniin inhibits the motility of SCC-9 and SCC-14 cells in vitro through a molecular mechanism that involves the attenuation of MMP-2 expression and activity mediated by decreased FAK/Src and ERK1/2 pathways.


Assuntos
Antineoplásicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glucosídeos/farmacologia , Taninos Hidrolisáveis/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Bucais/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Geranium/química , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/fisiopatologia , Quinases da Família src/genética
18.
Artif Cells Nanomed Biotechnol ; 47(1): 2641-2649, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31240956

RESUMO

Objective: Focal adhesion kinase (FAK) has critical functions in proliferation and differentiation of many cell types, however, the role of FAK on BMP9-induced osteogenic differentiation in SMSCs has not been characted. The purpose of current study is to explore the mechanism of FAK on the BMP9-induced osteogenesis of SMSCs in vitro and in vivo. Methods: The optimal dose of BMP9 was determined by incubation in different BMP9 concentrations, then cells were transfected with siRNA-induced FAK knockdown in BMP9-induced osteogenesis. Cell proliferation, migration, the osteogenic capacity, and the underlying mechanism were further detected in vitro. Imaging and pathological examination were conducted to observe the bone formation in vivo. Results: Our findings suggested that BMP9 could obviously promote FAK phosphorylation in osteogenic conditions. In contrast, FAK knockdown significantly decreased the cell proliferation, migration, the osteogenic capacity of SMSCs. To be specific, FAK knockdown could markedly inhibit the Wnt and MAPK signal pathway of SMSCs induced by BMP9. Besides, FAK knockdown could also effectively inhibit BMP-9-induced bone formation in vivo. Conclusion: FAK plays a pivotal role in promoting BMP9-induced osteogenesis of SMSCs, which is probably via activating Wnt and MAPK pathway.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/deficiência , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Membrana Sinovial/citologia
19.
Virology ; 534: 54-63, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176924

RESUMO

Influenza A virus (IAV) infections result in ∼500,000 global deaths annually. Host kinases link multiple signaling pathways at various stages of infection and are attractive therapeutic target. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, regulates several cellular processes including NFkB and antiviral responses. We investigated how FAK kinase activity regulates IAV pathogenesis. Using a severe infection model, we infected IAV-susceptible DBA/2 J mice with a lethal dose of H1N1 IAV. We observed reduced viral load and pro-inflammatory cytokines, delayed mortality, and increased survival in FAK inhibitor (Y15) treated mice. In vitro IAV-induced NFkB-promoter activity was reduced by Y15 or a dominant negative kinase-dead FAK mutant (FAK-KD) independently of the viral immune modulator, NS1. Finally, we observed reduced IAV-induced nuclear localization of NFkB in FAK-KD expressing cells. Our data suggest a novel mechanism where IAV hijacks FAK to promote viral replication and limit its ability to contribute to innate immune responses.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/enzimologia , NF-kappa B/imunologia , Provírus/patogenicidade , Animais , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos DBA , NF-kappa B/genética , Regiões Promotoras Genéticas , Provírus/genética , Provírus/fisiologia , Carga Viral , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Virulência , Replicação Viral
20.
Cell ; 177(7): 1738-1756.e23, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31104842

RESUMO

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.


Assuntos
Integrina beta1/metabolismo , Mecanotransdução Celular , Microdomínios da Membrana/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Cricetulus , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/genética , Microdomínios da Membrana/genética , Vinculina/genética , Vinculina/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
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