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1.
Reprod Biol Endocrinol ; 19(1): 70, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990206

RESUMO

Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a key role in contributing to the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the pathogenesis of ADS and puts an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with ß-estradiol (ß-E2) presented stronger proliferative and pro-angiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins. Meanwhile, these promoting effects were partially abrogated by Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of OV-Talin1 plus ß-E2 treatment. Results from the xenograft nude mice model showed that the hypodermic endometrial lesions from co-intervention group had the highest mean weight and volume, compared with that of individual OV-Talin1 or ß-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated the most in the co-intervention group. Our findings unveiled that overexpressed Talin1 might cooperate withß-E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.


Assuntos
Adenomiose/fisiopatologia , Endométrio/patologia , Células Estromais/fisiologia , Talina/fisiologia , Adenocarcinoma , Adenomiose/genética , Adenomiose/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Neoplasias do Endométrio , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Miométrio/patologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Organismos Livres de Patógenos Específicos , Células Estromais/efeitos dos fármacos , Talina/biossíntese , Talina/genética , Regulação para Cima
2.
Reprod Biol Endocrinol ; 19(1): 42, 2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750407

RESUMO

BACKGROUND: Endometriosis is a disease that involves active cell invasion and migration. Talin-1 can promote cell invasion, migration and adhension in various cancer cells, but its role in endometriosis has not been investigated. This study was to investigate the expression level of Talin-1 in endometriosis and the role of Talin-1 in the proliferation, adhesion, migration, and invasion of human endometrial stromal cells (ESCs). METHODS: Ectopic and eutopic endometrial tissues were collected from women with endometriosis, and the control endometrial tissues were obtained from patients without endometriosis. The expression level of Talin-1 was detected in each sample using quantitative real-time polymerase chain reaction and immunohistochemistry. The expression of Talin-1 was inhibited using RNA interference in ESCs, and its proliferation, apoptosis, adhesion, migration, and invasion capacity were analyzed. Western blotting was performed to detect the expression of related molecules after the downregulation of Talin-1. RESULTS: The results showed that the mRNA and protein expression of Talin-1 were significantly increased in the ectopic endometrium and eutopic endometrial tissues compared with the controls. The knockdown of Talin-1 did not affect the proliferation and apoptosis of ESCs. The results indicated that the downexpression of Talin-1 inhibited the adhesion, invasion, and migration of ESCs. In addition, the expressions of N-cadherin, MMP-2, and integrin ß3 were significantly lower after the deregulation of Talin-1, whereas the levels of E-cadherin were significantly increased. CONCLUSIONS: The expression of Talin-1 was increased in the ectopic and eutopic endometrial tissues compared with the control endometrium. The downregulation of Talin-1 inhibited the adhesion, invasion, and migration of ESCs.


Assuntos
Endometriose/genética , Endometriose/metabolismo , Endométrio/metabolismo , Talina/biossíntese , Proliferação de Células/fisiologia , Células Cultivadas , Endometriose/cirurgia , Endométrio/cirurgia , Feminino , Expressão Gênica , Humanos , Talina/genética
3.
PLoS One ; 11(3): e0152566, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27031101

RESUMO

INTRODUCTION: Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. RESULTS: The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3ß1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. CONCLUSION: In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.


Assuntos
Materiais Revestidos Biocompatíveis/química , Grafite/química , Membranas Artificiais , Nanoestruturas/química , Osteoblastos/metabolismo , Titânio/química , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Integrina alfa3beta1/biossíntese , Osseointegração , Osteoblastos/citologia , Paxilina/biossíntese , Talina/biossíntese
4.
BMC Cancer ; 16: 45, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26822056

RESUMO

BACKGROUND: Talin-1 (TLN-1) and TLN-2 are implicated in many cellular processes, but their roles in hepatocellular carcinoma (HCC) remain unclear. This study aimed to assess cell cycle distribution, anoikis, invasion and migration in human HCC MHCC-97 L cells. METHODS: MHCC-97 L cells, which highly express TLN-1, were transduced with TLN-1 shRNA (experimental group) or scramble shRNA (negative control group); non-transduced MHCC-97 L cells were used as blank controls. TLN-1 and TLN-2 mRNA and protein levels were detected by real-time RT-PCR and western blot, respectively. Then, cell cycle distribution and anoikis were assessed by flow cytometry. In addition, migration and invasion abilities were assessed using Transwell and cell scratch assays. Finally, a xenograft nude mouse model was established to further assess cell tumorigenicity. RESULTS: Compared with the blank and negative control groups, TLN-1/2 mRNA and protein levels were significantly reduced in the experiment group. TLN-1/2 knockdown cells showed significantly more cells in the G0/G1 phase (79.24%) in comparison with both blank (65.36%) and negative (62.69%) control groups; conversely, less cells were found in G2/M and S phases in the experimental group compared with controls. Moreover, anoikis was enhanced (P < 0.05), while invasion and migration abilities were reduced (P < 0.05) in TLN-1/2 knockdown cells compared with controls. TLN-1/2 knockdown inhibited MHCC-97 L cell migration (Percentage of wound healing area: experimental group: 32.6 ± 0.7% vs. negative controls: 50.1 ± 0.6% and blank controls: 53.6 ± 0.6%, both P < 0.01). Finally, the tumors obtained with TLN-1/2 knockdown cells were smaller (P < 0.05) compared with controls. CONCLUSION: Both TLN-1 and TLN-2 levels correlate with tumorigenicity in human HCC, indicating that these molecules constitute important molecular targets for the diagnosis and/or treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Talina/biossíntese , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Talina/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
BMC Cancer ; 15: 332, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25925041

RESUMO

BACKGROUND: Talin-1 is a cytoskeletal protein that plays an important role in tumourgenesis, migration and metastasis in several malignant tumors. The aim of this study was to evaluate the expression and prognostic value of Talin-1 in nasopharyngeal carcinoma (NPC). METHODS: Talin-1 mRNA and protein expression were examined in NPC cell lines and clinical nasopharyngeal tissues by quantitative RT-PCR, agarose gel electrophoresis and western blotting. The expression of Talin-1 was analyzed by immunohistochemical staining in 233 paraffin-embedded NPC specimens with clinical follow-up data and cox regression analysis was used to identify independent prognostic factors. The functional role of Talin-1 in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by the wound healing and transwell invasion assays. RESULTS: The expression of Talin-1 was significantly upregulated in most NPC cell lines and clinical tissues at both the mRNA and protein levels. High expression of Talin-1 was significantly associated with distant metastasis (P = 0.001) and patient death (P = 0.001). In addition, high expression of Talin-1 was associated with significantly poorer overall survival (OS: HR, 2.15; 95% CI, 1.28-3.63; P = 0.003) and poorer distant metastasis-free survival (DMFS: HR, 2.39; 95% CI, 1.38-4.15; P = 0.001). Cox regression analysis indicated that high expression of Talin-1 and TNM stage were independent prognostic indicators (both P < 0.05). Stratified analysis demonstrated that high expression of Talin-1 was associated with significantly poorer survival in patients with advanced stage disease (stage III-IV, HR, 1.91; 95% CI, 1.09-3.35; P = 0.02 for OS and HR, 2.22; 95% CI, 1.24-3.99; P = 0.006 for DMFS). Furthermore, the depletion of Talin-1 suppressed the migratory and invasive ability of NPC cells in vitro. CONCLUSIONS: Our data demonstrate that high expression of Talin-1 is associated with significantly poorer OS and poorer DMFS in NPC and depletion of Talin-1 expression inhibited NPC cell migration and invasion. Talin-1 may serve as novel prognostic biomarker in NPC.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Nasofaríngeas/genética , Prognóstico , Talina/biossíntese , Idoso , Biomarcadores Tumorais/genética , Carcinoma , Linhagem Celular Tumoral , Movimento Celular/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , RNA Mensageiro/biossíntese , Talina/genética
6.
Histol Histopathol ; 30(4): 465-72, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25371229

RESUMO

Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.


Assuntos
Adaptação Fisiológica/fisiologia , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Tendões/metabolismo , Animais , Colágeno Tipo IV/biossíntese , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Imunofluorescência , Imuno-Histoquímica , Masculino , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Talina/biossíntese , Vinculina/biossíntese
7.
Oncogene ; 34(14): 1811-21, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-24793790

RESUMO

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that ß1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how ß1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of ß1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in ß1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of ß1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased ß1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent ß1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to ß1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Ósseas/secundário , Proteínas de Ciclo Celular/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Integrina beta1/metabolismo , Talina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Anoikis/genética , Adesão Celular , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Adesões Focais/metabolismo , Humanos , Metástase Linfática , Masculino , Camundongos , Fosforilação , Próstata/patologia , Neoplasias da Próstata/patologia , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Talina/biossíntese , Talina/genética
8.
Eur Rev Med Pharmacol Sci ; 18(17): 2533-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25268101

RESUMO

OBJECTIVE: Cell adhesion and angiogenesis within the extracellular matrix involve special signaling molecules, such as integrins and the actin binding protein Talin-1. The aim of this study was to investigate and describe the expression of Talin-1 for the early detection of colon cancer. PATIENTS AND METHODS: Blood serum samples were collected from 50 healthy volunteers and from 90 patients with colon cancer. Using an enzyme-linked immunosorbent assay (ELISA), all 140 samples were analyzed. RESULTS: Preoperative levels of Talin-1 in the serum were significantly higher in patients with colon cancer (p < 0.001). No significant correlation was found between preoperative levels of Talin-1 in the serum and the age and gender of the patients (p < 0.05). However, a significant correlation was found between Talin-1 levels and the tumor grade, TNM stage, and lymph node metastasis (p < 0.001). CONCLUSIONS: Talin-1 may play a role in the reinforcement of cell proliferation, cell adhesion, and angiogenesis in colon cancer. Thus, the Talin-1 protein activity may be a novel biomarker to detect colon cancer in humans.


Assuntos
Neoplasias do Colo/sangue , Talina/sangue , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Adesão Celular/fisiologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/patologia , Detecção Precoce de Câncer/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Talina/biossíntese
9.
PLoS One ; 9(5): e98208, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24858105

RESUMO

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias Experimentais/metabolismo , Talina/biossíntese , Neoplasias da Língua/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Movimento Celular/genética , Adesões Focais/genética , Adesões Focais/patologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteômica/métodos , Talina/genética , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Regulação para Cima/genética
10.
FASEB J ; 28(6): 2551-65, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24604078

RESUMO

Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.


Assuntos
Barreira Hematoencefálica/fisiologia , MicroRNAs/fisiologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Humanos , Masculino , Camundongos , Esclerose Múltipla , Talina/biossíntese , Transcriptoma , Regulação para Cima , Vinculina/biossíntese
11.
Cell Signal ; 26(7): 1489-99, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24662262

RESUMO

Syndecans are cell membrane proteoglycans that can modulate the activity and dynamics of some growth factor receptors and integrins. Here, we show the down-regulation of integrin lymphocyte function-associated antigen-1 (LFA-1) and inhibition of adhesion of Jurkat T cells transfected with syndecan-2. The PDZ-binding domain in the cytoplasmic region of syndecan-2 was necessary to block the LFA-1 high-affinity conformation, and to reduce cellular adhesion. A second cytoplasmic motif comprising tyrosines 179 and 191, and serines 187 and 188 contributed also to reduce LFA-1 function and cellular adhesion. Inhibition of the LFA-1 high-affinity conformation by syndecan-2 was independent of the expression of the talin head domain and RhoA, Rac1 and Cdc42 GTPases. These results demonstrate the importance of PDZ-binding domain of syndecan-2 for controlling LFA-1 affinity and cell adhesion.


Assuntos
Adesão Celular/fisiologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Domínios PDZ/genética , Sindecana-2/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Regulação para Baixo , Endotélio/citologia , Endotélio/metabolismo , Proteínas Ativadoras de GTPase/biossíntese , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfoproteínas/biossíntese , Ligação Proteica , Sindecana-2/genética , Linfócitos T/metabolismo , Talina/biossíntese , Transfecção , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese
12.
J Biol Chem ; 288(6): 4252-64, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23266827

RESUMO

Integrins are adhesive, signaling, and mechanotransduction proteins. Talin (Tln) activates integrins and links it to the actin cytoskeleton. Vertebrates contain two talin genes, tln1 and tln2. How Tln1 and Tln2 function in cardiac myocytes (CMs) is unknown. Tln1 and Tln2 expression were evaluated in the normal embryonic and adult mouse heart as well as in control and failing human adult myocardium. Tln1 function was then tested in the basal and mechanically stressed myocardium after cardiomyocyte-specific excision of the Tln1 gene. During embryogenesis, both Tln forms are highly expressed in CMs, but in the mature heart Tln2 becomes the main Tln isoform, localizing to the costameres. Tln1 expression is minimal in the adult CM. With pharmacological and mechanical stress causing hypertrophy, Tln1 is up-regulated in CMs and is specifically detected at costameres, suggesting its importance in the compensatory response to CM stress. In human failing heart, CM Tln1 also increases compared with control samples from normal functioning myocardium. To directly test Tln1 function in CMs, we generated CM-specific Tln1 knock-out mice (Tln1cKO). Tln1cKO mice showed normal basal cardiac structure and function but when subjected to pressure overload showed blunted hypertrophy, less fibrosis, and improved cardiac function versus controls. Acute responses of ERK1/2, p38, Akt, and glycogen synthase kinase 3 after mechanical stress were strongly blunted in Tln1cKO mice. Given these results, we conclude that Tln1 and Tln2 have distinct functions in the myocardium. Our data show that reduction of CM Tln1 expression can lead to improved cardiac remodeling following pressure overload.


Assuntos
Cardiomegalia/metabolismo , Miocárdio/metabolismo , Talina/biossíntese , Adulto , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Feminino , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Knockout , 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 , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Fisiológico/genética , Talina/genética , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
J Biol Chem ; 287(17): 13799-812, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22351767

RESUMO

Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482-787)), and the THD restrains talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the talin rod domain that regulate the subcellular localization of talin.


Assuntos
Regulação da Expressão Gênica , Talina/biossíntese , Actinas/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Citosol/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Frações Subcelulares/metabolismo , Talina/química
14.
PLoS Biol ; 9(12): e1001223, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22205879

RESUMO

Mechanotransduction is a critical function for cells, in terms of cell viability, shaping of tissues, and cellular behavior. In vitro, cellular level forces can stretch adhesion proteins that link extracellular matrix to the actin cytoskeleton exposing hidden binding sites. However, there is no evidence that in vivo forces produce significant in vivo stretching to cause domain unfolding. We now report that the adhesion protein, talin, is repeatedly stretched by 100-350 nm in vivo by myosin contraction of actin filaments. Using a functional EGFP-N-Talin1-C-mCherry to measure the length of single talin molecules, we observed that the C-terminal mCherry was normally displaced in the direction of actin flow by 90 to >250 nm from N-EGFP but only by 50-60 nm (talin's length in vitro) after myosin inhibition. Individual talin molecules transiently stretched and relaxed. Peripheral, multimolecular adhesions had green outside and red proximal edges. They also exhibited transient, myosin-dependent stretching of 50-350 nm for 6-16 s; however, expression of the talin-binding head of vinculin increased stretching to about 400 nm and suppressed dynamics. We suggest that rearward moving actin filaments bind, stretch, and release talin in multiple, stochastic stick-slip cycles and that multiple vinculin binding and release cycles integrate pulling on matrices into biochemical signals.


Assuntos
Mecanotransdução Celular , Talina/biossíntese , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Linhagem Celular , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Talina/metabolismo
15.
J Pathol ; 224(3): 367-76, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21547905

RESUMO

Oral squamous cell carcinoma (OSCC) is highly invasive and is associated with frequent tumour recurrences and lymph node metastases. Identification of genes involved in the aggressiveness of OSCC may provide new targets for clinical intervention. A genome-wide study based on the Sty1 250K SNP array indicated the involvement of the Talin-1 (TLN1) gene in the 9p13.3 amplicon, which was further validated by dual colour fluorescence in situ hybridization (FISH). Comparative analyses revealed that TLN1 was the most highly expressed integrin-cytoskeleton cross-linker that can trigger integrin activation. IHC analyses and mouse study also revealed an association between TLN1 overexpression and advanced OSCC with invasion to adjacent tissues. Survival analyses indicated a significant association between TLN1 genetic gain/overexpression and a reduced overall survival in patients. Functional knockdown by a dominant negative TLN1 fragment reduced cell growth and invasiveness in TLN1-overexpressing cells via inactivation of downstream oncogenic signalling. The present study suggests an important role for TLN1 in oral cancer development. TLN1 overexpression could serve as a diagnostic marker for aggressive phenotypes and a potential target for treating OSCC.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundário , Neoplasias Bucais/metabolismo , Talina/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Prognóstico , Análise de Sobrevida , Talina/genética , Células Tumorais Cultivadas
16.
J Urol ; 185(6): 2314-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21511284

RESUMO

PURPOSE: Ureteropelvic junction obstruction is one of the most common causes of hydronephrosis in children. A malfunction of smooth muscle cells is believed to be the underlying mechanism causing obstruction. We investigated the expression of some integrins, talin and ß-dystroglycan, considered the main compound of smooth muscle cell cytoskeleton, and active caspase 3 at the level of the ureteropelvic junction obstruction. MATERIALS AND METHODS: Specimens were obtained at pyeloplasty in 12 children with ureteropelvic junction obstruction. Six control specimens were obtained during organ explantation. Specimens were divided into renal pelvis, ureteropelvic junction and ureter below the obstruction. Western blot analysis of active caspase 3, and immunofluorescence and polymerase chain reaction analysis were performed for α7A, ß1A, α7B and ß1D integrins, talin and ß-dystroglycan. RESULTS: Talin and ß-dystroglycan were slightly impaired in ureteropelvic junction obstruction, while α7B and ß1D integrins were severely reduced, and α7A, ß1A and active caspase 3 were significantly enhanced compared to controls. CONCLUSIONS: We demonstrated activation of apoptosis and a critical alteration of cytoskeleton that might explain the altered function and the increased apoptosis in smooth muscle cells in ureteropelvic junction obstruction. The delayed rearrangement of the cytoskeleton of smooth muscle cells in ureteropelvic junction obstruction might be linked to a postnatal splicing from α7A and ß1A to α7B and ß1D integrins, respectively. This relationship could explain the common clinical scenario of spontaneous improvement of hydronephrosis in children with suspected ureteropelvic junction obstruction.


Assuntos
Citoesqueleto/ultraestrutura , Pelve Renal/patologia , Músculo Liso/patologia , Obstrução Ureteral/patologia , Caspase 3/biossíntese , Pré-Escolar , Distroglicanas/biossíntese , Humanos , Imuno-Histoquímica , Lactente , Integrinas/biossíntese , Talina/biossíntese , Obstrução Ureteral/metabolismo
17.
Cancer Res ; 70(5): 1885-95, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20160039

RESUMO

Talin1 is a focal adhesion complex protein that regulates integrin interactions with ECM. This study investigated the significance of talin1 in prostate cancer progression to metastasis in vitro and in vivo. Talin1 overexpression enhanced prostate cancer cell adhesion, migration, and invasion by activating survival signals and conferring resistance to anoikis. ShRNA-mediated talin1 loss led to a significant suppression of prostate cancer cell migration and transendothelial invasion in vitro and a significant inhibition of prostate cancer metastasis in vivo. Talin1-regulated cell survival signals via phosphorylation of focal adhesion complex proteins, such as focal adhesion kinase and Src, and downstream activation of AKT. Targeting AKT activation led to a significant reduction of talin1-mediated prostate cancer cell invasion. Furthermore, talin1 immunoreactivity directly correlated with prostate tumor progression to metastasis in the transgenic adenocarcinoma mouse prostate mouse model. Talin1 profiling in human prostate specimens revealed a significantly higher expression of cytoplasmic talin1 in metastatic tissue compared with primary prostate tumors (P < 0.0001). These findings suggest (a) a therapeutic significance of disrupting talin1 signaling/focal adhesion interactions in targeting metastatic prostate cancer and (b) a potential value for talin1 as a marker of tumor progression to metastasis.


Assuntos
Adenocarcinoma/patologia , Anoikis/fisiologia , Adesões Focais/fisiologia , Neoplasias da Próstata/patologia , Talina/fisiologia , Adenocarcinoma/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Talina/biossíntese
18.
J Exp Med ; 204(13): 3113-8, 2007 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-18086864

RESUMO

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin beta subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.


Assuntos
Plaquetas/citologia , Integrinas/metabolismo , Agregação Plaquetária , Talina/biossíntese , Trombose/metabolismo , Animais , Arteríolas/metabolismo , Plaquetas/metabolismo , Cloretos , Citoplasma/metabolismo , Compostos Férricos/farmacologia , Citometria de Fluxo/métodos , Camundongos , Camundongos Transgênicos , Microscopia/métodos , Modelos Genéticos , Adesividade Plaquetária
19.
Microvasc Res ; 64(1): 47-55, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12074630

RESUMO

The microfibrils of anchoring filaments, a typical ultrastructural feature of initial lymphatic vessels, consist mainly of fibrillin and are similar to the microfibrils of elastic fibers. As we previously demonstrated, they radiate from focal adhesions of lymphatic endothelium to the perivascular elastic network. Although present in large blood vessels, fibrillin microfibrils have never been detected in blood capillaries. Here we report immunohistochemical evidence that cultured bovine aortic and lymphatic endothelial cells express fibrillin microfibrils. These microfibrils form an irregular web in lymphatic endothelial cells, whereas in blood vessel endothelial cells they are arranged in a honeycomb pattern. Cultured lymphatic and blood vessel endothelial cells also produce focal adhesion molecules: focal adhesion kinase, vinculin, talin, and cytoskeletal beta-actin. Our data suggest that anchoring filaments of initial lymphatic vessels in vivo may be produced by endothelium. Through their connection with focal adhesions, they may form a mechanical anchorage for the thin wall of initial lymphatic vessels and a transduction device for mechanical signals from the extracellular matrix into biochemical signals in endothelial cells. The complex anchoring filaments-focal adhesions may control the permeability of lymphatic endothelium and finely adjust lymph formation to the physiological conditions of the extracellular matrix. The different deposition of fibrillin microfibrils in blood vessel endothelial cells may be related to the necessity of withstanding shear forces. Thus, in our opinion, differences in fibrillin deposition imply a different role of fibrillin in blood vessel and lymphatic endothelium.


Assuntos
Actinas/biossíntese , Endotélio Vascular/citologia , Sistema Linfático/citologia , Proteínas dos Microfilamentos/biossíntese , Proteínas Tirosina Quinases/biossíntese , Talina/biossíntese , Vinculina/biossíntese , Animais , Bovinos , Células Cultivadas , Citoplasma/metabolismo , Fibrilinas , Proteína-Tirosina Quinases de Adesão Focal , Imuno-Histoquímica , Ducto Torácico/citologia , Fatores de Tempo
20.
Biochem Biophys Res Commun ; 286(5): 880-5, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11527381

RESUMO

We have utilised genomic and EST databases to assemble the sequence of the human talin2 (TLN2) gene. Talin2 protein is similar in size and sequence to talin1 throughout its length (74% identity, 86% similarity). The major differences are in (i) the size of the genes, the TLN2 gene is >200 kb compared with approximately 30 kb for TLN1 due to a difference in intron size, although intron/exon boundaries, with the exception of two, are strictly conserved; (ii) the expression patterns, TLN1 gives rise to an approximately 8-kb mRNA which is observed in all tissues, whereas TLN2 gives rise to multiple transcripts with the highest levels in heart.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Talina/química , Talina/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 9 , Sequência Conservada , Proteínas do Citoesqueleto/biossíntese , Éxons , Etiquetas de Sequências Expressas , Humanos , Íntrons , Dados de Sequência Molecular , Miocárdio/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Talina/biossíntese , Distribuição Tecidual
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