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1.
Int Immunol ; 35(7): 339-348, 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37083755

RESUMO

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin-like transcript 3/leukocyte immunoglobulin-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B was identified very recently as fibronectin, particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind fibronectin on target cells in trans together with integrins, classical fibronectin receptors, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrins suggested that these novel and classical fibronectin receptors, respectively, co-engage fibronectin immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized fibronectin. While surface fibronectin-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of fibronectin-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing fibronectin on target cells, which sheds light on the unexpected role of LILRB4 and fibronectin as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.


Assuntos
Fibronectinas , Células Matadoras Naturais , Camundongos , Animais , Humanos , Fibronectinas/metabolismo , Integrinas/metabolismo , Receptores de Fibronectina/metabolismo , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo
2.
Tohoku J Exp Med ; 257(3): 171-180, 2022 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-35691913

RESUMO

A myeloid immune checkpoint, leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/CD85k in humans and gp49B in mice) is expressed on dendritic cells (DCs). However, a mode of regulation of DCs by B4/gp49B is not identified yet in relation to the ligand(s) as well as to the counteracting, activation-type receptor. Our recent identification of the physiological/pathological ligand for B4/gp49B as the fibronectin (FN) N-terminal 30-kDa domain poses the question of the relationship between B4/gp49B and a classical FN receptor/cellular activator, integrin, on DCs. Here we showed that FN is not constitutively tethered on the surface of bone marrow-derived cultured DCs (BMDCs) or splenic DCs, even though the FN receptor integrin and gp49B are co-expressed on these cells. Confocal laser scanning microscopic analysis, however, revealed weak correlation of fluorescent signals between gp49B and integrin ß1, suggesting their partial co-localization on the BMDC surface even in the absence of FN. We found that the plating of BMDCs onto immobilized FN induced tyrosine phosphorylation of focal adhesion kinase (FAK) and spleen tyrosine kinase (Syk). In the absence of gp49B, while the FAK phosphorylation level was virtually unchanged, that of phosphorylation of Syk was markedly augmented. These results suggested that the immobilized FN induced a crosstalk between gp49B and integrin in terms of the intracellular signaling of BMDCs, in which gp49B suppressed the integrin-mediated pro-inflammatory cascade. Our observations may provide a clue for elucidating the mechanism of the therapeutic efficacy of B4/gp49B blocking in autoimmune disease and cancer.


Assuntos
Integrinas , Receptores de Fibronectina , Animais , Adesão Celular , Células Dendríticas/metabolismo , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrinas/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo , Camundongos , Fosforilação , Receptores de Fibronectina/metabolismo , Receptores Imunológicos/metabolismo
3.
Cells ; 9(12)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33353083

RESUMO

Fibronectin is a ubiquitous extracellular matrix protein that is produced by many cell types in the bone marrow and distributed throughout it. Cells of the stem cell niche produce the various isoforms of this protein. Fibronectin not only provides the cells a scaffold to bind to, but it also modulates their behavior by binding to receptors on the adjacent hematopoietic stem cells and stromal cells. These receptors, which include integrins such as α4ß1, α9ß1, α4ß7, α5ß1, αvß3, Toll-like receptor-4 (TLR-4), and CD44, are found on the hematopoietic stem cell. Because the knockout of fibronectin is lethal during embryonal development and because fibronectin is produced by almost all cell types in mammals, the study of its role in hematopoiesis is difficult. Nevertheless, strong and direct evidence exists for its stimulation of myelopoiesis and thrombopoiesis using in vivo models. Other reviewed effects can be deduced from the study of fibronectin receptors, which showed their activation modifies the behavior of hematopoietic stem cells. Erythropoiesis was only stimulated under hemolytic stress, and mostly late stages of lymphocytic differentiation were modulated. Because fibronectin is ubiquitously expressed, these interactions in health and disease need to be taken into account whenever any molecule is evaluated in hematopoiesis.


Assuntos
Fibronectinas/fisiologia , Hematopoese , Receptores de Fibronectina/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Eritropoese , Células-Tronco Hematopoéticas/citologia , Hemólise , Humanos , Receptores de Hialuronatos/metabolismo , Integrinas/metabolismo , Camundongos , Mielopoese , Nicho de Células-Tronco , Células-Tronco/citologia , Trombopoese , Receptor 4 Toll-Like/metabolismo
4.
Talanta ; 212: 120718, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113526

RESUMO

Currently, fluorescent imaging is one of the most promising diagnostic approaches for facile detection of cancers in situ in thanks to a fluorescent probe. Two novel polypeptide-based fluorescent probes for different biomarkers to cancers are reported here. These probes focused on tyrosine-isoleucine-glycine-serine-arginine (YIGSR) and arginine-glycine-aspartic (RGD), which receptors play an important role in the extracellular matrix and are overexpressed in tumor cells and then can be used as tumor-targeting groups in fluorescent imaging. In this work, the pentpeptide-rhodamine B derivative (YIGSR-RhB) and tripeptide-rhodamine B derivative (RGD-RhB) were synthesized respectively by using the solid phase synthesis methods. These derivatives were further characterized by 1HNMR, MS, UV and IR, etc. Their fluorescent and biocompatibility properties, such as the cell cytotoxicity, cell uptake and fluorescent imaging of tumor cells, and fluorescent imaging in BALB/c female mice with 4T1 tumors and C57 mice with B16F10 tumor in vivo, were also measured. Experiment results demonstrated that YIGSR-RhB and RGD-RhB possessed the low cell cytotoxicity, good tumor-targeting property and fluorescent properties similar to rhodamine B. Moreover, YIGSR-RhB and RGD-RhB can be taken up highly by the B16F10 melanoma cells and 4T1 breast cancer cells, and then achieve the good fluorescent imaging in these tumor cells in vitro and tumors of mice in vivo. Therefore, YIGSR-RhB and RGD-RhB can be used as the potential tumor-targeting probes for fluorescent imaging. They can directly attach the cell membrane and specifically target to the tumor cells.


Assuntos
Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Oligopeptídeos/química , Rodaminas/química , Animais , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Laminina/química , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Oligopeptídeos/síntese química , Oligopeptídeos/toxicidade , Imagem Óptica , Receptores de Fibronectina/química , Rodaminas/síntese química , Rodaminas/toxicidade
6.
Zhonghua Bing Li Xue Za Zhi ; 46(3): 182-186, 2017 Mar 08.
Artigo em Chinês | MEDLINE | ID: mdl-28297759

RESUMO

Objective: To investigate the expression of integrin α5ß1 and fibronectin in the human aorta and coronary artery, and their effects in the development of atherosclerosis. Methods: One hundred and twenty autopsy aorta and coronary artery specimens were collected, and the expression of CD68, actin, integrin α5ß1 and fibronectin was detected by immunohistochemical staining. Atherosclerotic plaques were located by CD68 and actin staining, and the degree of coronary artery stenosis was determined by elastic fiber staining and NIH Scion Image(60.1) software. The coronary artery tissues were divided into groups A (0-25%); B (26%-50%); C (51%-75%) and D (76%-100%) according to the degree of stenosis. Results: Integrin α5ß1 showed cytoplasmic expression in endothelium, foam cells, monocytes, smooth muscle cells and adjacent tissue around calcification. In both the aorta and coronary artery, integrin α5ß1 expression was stronger in the smooth muscle cells in the internal elastic lamina than in the tunica. The expression intensity in coronary artery smooth muscle decreased with increasing degree of coronary artery stenosis. Fibronectin showed cytoplasmic expression in foam cells, monocytes, smooth muscle cells of the internal elastic lamina and adjacent tissue around calcification. There was positive correlation of fibronectin and integrin α5ß1 expression in smooth muscle cells and adjacent tissue around calcification. Conclusions: In the development of atherosclerosis, integrin α5ß1 and fibronectin may participate in the regulating the migration of smooth muscle cells to the intima, and promote the formation of local calcification of atherosclerotic plaques. But integrin α5ß1 is not involved in the late stage of atherosclerosis with increasing coronary artery stenosis.


Assuntos
Aorta/patologia , Vasos Coronários/patologia , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/patologia , Actinas/metabolismo , Aorta/metabolismo , Autopsia , Constrição Patológica , Vasos Coronários/metabolismo , Endotélio Vascular , Humanos , Imuno-Histoquímica , Músculo Liso Vascular/patologia , Receptores de Fibronectina/metabolismo , Túnica Íntima
7.
Araçatuba; s.n; 2017. 61 p. tab, ilus.
Tese em Inglês, Português | BBO - Odontologia | ID: biblio-881466

RESUMO

Objetivo: Avaliar a resposta tecidual e a capacidade de biomineralização dos materiais endodônticos SK Seal Root Canal Sealer (SK Seal), Sealer 26® e AH plus® em tecido subcutâneo de ratos. Material e Métodos: Vinte e quatro ratos Wistar (n=6) receberam implantes subcutâneo contendo os cimentos e um tubo vazio como controle. Após 7, 15, 30 e 60 dias, os animais foram eutanasiados e os tubos de polietileno foram removidos junto com o tecido circunjacente. Em seguida, os espécimes foram processados para análise em Hematoxilina-Eosina, von Kossa, luz polarizada e imunoistoquímica para fibronectina (FN) e tenascina (TN). Os dados foram tabulados e analisados através do teste de Kruskal-Wallis e Dunn (p<0,05). Resultados: Todos os materiais testados induziram uma reação inflamatória moderada aos 7 e 15 dias (p> 0,05). Não foram observadas diferenças entre os grupos após 30 ou 60 dias (p> 0,05). A cápsula fibrosa foi considerada espessa aos 7 dias, tornando-se fina no final do experimento. Todos os grupos apresentaram marcadores positivos para FN e TN em todos os tempos de análise, com maior imunomarcação para os cimentos em comparação ao grupo controle (p <0,05). Os cimentos não apresentaram von Kossa positiva ou estruturas birrefringentes à luz polarizada. Conclusão: Todos os cimentos testados apresentaram biocompatibilidade, porém não estimularam a mineralização(AU)


Aim: The aim of this study was to evaluate biocompatibility and biomineralization of the endodontic materials SK Seal Root Canal Sealer (SK Seal), Sealer 26® and AH plus® in subcutaneous tissue of rats. Methodology: Twenty-four Wistar rats (n=6) received subcutaneous implants containing the test sealers, and an empty tube as control. After 7, 15, 30 and 60 days, the animals were killed and polyethylene tubes were removed with the surrounding tissues. The pieces were processed for Hematoxylin-Eosin, von Kossa, polarized light and immunohistochemical analysis for fibronectin (FN) and tenascin (TN). Data were tabulated and analyzed via Kruskal-Wallis and Dunn's test (p<0,05). Results: All tested materials induced a moderate infammatory reaction after 7 and 15 days. (p>0,05). No difference was observed among groups after days 30 or 60 days (p>0.05). The fibrous capsule was considered thick on the 7th day, and classified as thin at the end of the experiment. All groups presented positive markers for FN and TN in all analyzed time, with higher immunolabeling to sealers in comparison with the control group (p<0,05). The sealers did not present von Kossa positive or birefringent structures to polarized light. Conclusion: All tested sealers demonstrated biocompatibility, but did not stimulate the mineralization(AU)


Assuntos
Animais , Ratos , Inflamação , Obturação do Canal Radicular , Calcificação Fisiológica , Ratos Wistar , Receptores de Fibronectina , Tenascina
8.
J Microbiol Immunol Infect ; 49(2): 249-56, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25081983

RESUMO

BACKGROUND/PURPOSE: Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. METHODS: Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5ß1 and α4ß1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. RESULTS: Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4ß1 integrin. An increased expression level of α4ß1 integrin in comparison with α5ß1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. CONCLUSION: Increased expression level of α4ß1 in differentiated THP-1 cells could suggest the important role of α4ß1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Fibronectinas/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiologia , Receptores de Fibronectina/análise , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Integrina alfa4beta1/análise , Integrina alfa4beta1/genética , Integrina alfa5beta1/análise , Integrina alfa5beta1/genética , Mycobacterium tuberculosis/genética , Ligação Proteica , Receptores de Fibronectina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Stem Cells Dev ; 22(16): 2315-25, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23517131

RESUMO

An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration after intracardiac transplantation. Since nothing has been reported as to the factors controlling hESC-CM migration, we hypothesized that hESC-CMs would migrate in response to the extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live-cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly a fivefold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins α-5 and α-V. Next, we screened 10 soluble candidate factors by transwell assay and found that the noncanonical Wnt, Wnt5a, elicited an approximately twofold increase in migration over controls. This effect was confirmed using the gap-closure assay, in which Wnt5a-treated hESC-CMs showed approximately twofold greater closure than untreated cells. Studies with microfluidic-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust promigratory responses to FN and Wnt5a, findings that have implications on both cardiac development and cell-based therapies.


Assuntos
Células-Tronco Embrionárias/citologia , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Wnt/farmacologia , Anticorpos Neutralizantes/farmacologia , Adesão Celular , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Cultura em Câmaras de Difusão , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Imagem Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fibronectina/antagonistas & inibidores , Receptores de Fibronectina/genética , Receptores de Fibronectina/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a
10.
J Endocrinol Invest ; 36(6): 375-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23027776

RESUMO

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by the presence of specific antibodies and by a lymphocytic infiltration of the thyroid secreting inflammatory cytokines. Macrophages, lymphocytes, and cytokines play a pivotal role in both development and progression of Th1-mediated autoimmune diseases, and a direct role in the destruction of thyroid follicles and follicular cell function in autoimmune thyroiditis. Integrins are integral membrane receptors involved in cell-extra-cellular matrix (ECM) interaction with both structural and signaling functions. The integrin- ECM interaction is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of cytokine stimulation on integrin expression and signaling in the thyroid cell. Primary cultures from normal thyroids were treated with interferon-γ (IFN-γ), INF-α, tumor necrosis factor-α, interleukin 1a or these cytokines all together. Integrin expression, cell adhesion to fibronectin (FN) and FN-stimulated extracellular signal-regulated kinase (ERK) phosphorylation were determined after cytokine treatment. IFN-γ and IFN-α were the most effective, reducing the expression of the integrin αvß3 and slightly increasing the α3ß1. Cell treatment with IFN-γ strongly impaired cell adhesion to FN. At the same time, the treatment with IFN-γ dramatically inhibited the stimulation of ERK phosphorylation induced by cell adhesion to FN. In conclusion, IFN-γ inhibits the expression of the integrin αvß3, reducing the cell adhesion to FN and the following intracellular signaling in thyroid cells in culture. These results suggest that integrins may be a target of the infiltrating lymphocytes and have a role in the pathogenesis of autoimmune thyroiditis.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Integrinas/fisiologia , Interferon gama/farmacologia , Glândula Tireoide/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Citocinas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fibronectinas/farmacologia , Fibronectinas/fisiologia , Humanos , Integrinas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Fibronectina/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiologia , Células Tumorais Cultivadas
11.
Pathol Biol (Paris) ; 60(1): 15-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22265966

RESUMO

In this review, we present several aspects of cell-matrix interactions, especially the role of fibronectin and integrins in the mediation of these interactions. As this field of investigations literally exploded over the last decades, we had to limit this review to some aspects of this field. We cited experiments giving details on the modifications of fibronectin molecules during their interactions with cells as well as on recent progress of the molecular mechanisms of fibronectin-integrin interactions. We insisted on the molecular details which were shown to play a role in the bi-directional signals "sent" by cells to the surrounding matrix (inside-out and outside-in). A number of recent publications confirmed the physiopathological importance of these messages both for the normal function of tissues as well as for the understanding of their pathological modifications. We insist also on the importance of fibronectin-fragments during some pathologies.


Assuntos
Comunicação Celular/fisiologia , Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Integrinas/fisiologia , Animais , Coleta de Dados , Matriz Extracelular/metabolismo , Fibronectinas/sangue , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Fenômenos Fisiológicos da Nutrição , Receptores de Fibronectina/genética , Receptores de Fibronectina/metabolismo , Receptores de Fibronectina/fisiologia
12.
Int J Oncol ; 39(2): 393-400, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21567080

RESUMO

We have previously shown that the genotoxin-induced apoptosis in mouse embryo fibroblasts was enhanced by the extracellular matrix protein fibronectin (FN). In the present study, we tested the hypothesis that FN regulates the DNA damage response (DDR) signaling pathways in HCT116 (p53-wt) and HT29 (p53-mut) human colon cancer cells and tumor-derived myofibroblasts. DNA damage recognition mechanisms were analyzed by immunofluorescence staining, cell cycle analysis and immunoblotting addressed at specific molecular sensors and executors involved in the DDR pathways. The results show that FN, but not collagen type IV or Matrigel, initiates and potentiates the DDR to the anticancer drug cisplatin in an α5 integrin and cell cycle-dependent manner (S and G2/M phases) in human colon cancer cells. Accordingly, we demonstrate that adhesion of HCT116 cells to FN upregulated the expression of α5 integrin FN receptors at the cell surface. These FN-induced DDR pathways include the concerted phosphorylation of histone H2AX on Ser139 detected as nuclear foci (γ-H2AX, 15 and 25 kDa forms), of ataxia telangiectasia mutated (ATM-Ser1981), checkpoint kinase 2 (CHK2-Thr68, 62 and 67 kDa) and p53-Ser15. These FN-induced γ-H2AX signals were interrupted or attenuated by selective inhibitors acting on the DDR pathway kinases, including wortmannin (targeting the phosphatidylinositol-3-kinase-related protein kinases, PIKK), KU55933 (ATM), NU7026 (DNA-dependent protein kinase catalytic subunit, DNA-PK-cs) and SP600125 (JNK2, stress activated protein kinase/c-Jun N-terminal kinase-2). Adhesion to FN also potentiated the γ-H2AX signals and the cytotoxic effects of cisplatin in human colon tumor-derived myofibroblasts. These data provide evidence that FN promotes DNA damage recognition and chemosensitization to cisplatin via the potentiation of the DNA damage signaling responses in human colon cancer cells and tumor-derived myofibroblasts.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Miofibroblastos/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase do Ponto de Checagem 2 , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , Miofibroblastos/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fibronectina/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Cell Death Differ ; 18(5): 806-16, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21113146

RESUMO

Anoikis resistance is a hallmark of transformed epithelial cells. Here, we show that treatment of anoikis-resistant carcinoma cell lines with the endogenous lectin galectin-1 (Gal-1) promoted apoptosis via interaction with the unligated fibronectin receptor α(5)ß(1)-integrin. Gal-1 efficiency correlated with expression of α(5)ß(1)-integrin, and transfection of the α(5)-subunit into deficient cell lines conferred Gal-1 binding and anoikis stimulation. Furthermore, Gal-1 and the α(5)- and ß(1)-integrin subunits co-precipitated in Gal-1-stimulated cells undergoing anoikis. Other members of the galectin family failed to be active. The functional interaction between Gal-1 and α(5)ß(1)-integrin was glycan dependent with α2,6-sialylation representing a switch-off signal. Desialylation of cell surface glycans resulted in increased electrophoretic mobility of α(5)ß(1)-integrin and facilitated Gal-1 binding and anoikis stimulation. On the level of signaling, Gal-1-stimulated anoikis was prevented by filipin, which impaired the internalization of α(5)ß(1)-integrin via cholesterol-enriched microdomains, and by pretreatment with a caspase-8 inhibitor. We propose that Gal-1/α(5)ß(1)-integrin interaction participates in the control of epithelial integrity and integrin sialylation may enable carcinoma cells to evade this Gal-1-dependent control mechanism.


Assuntos
Anoikis , Caspase 8/metabolismo , Galectina 1/fisiologia , Integrina alfa5beta1/metabolismo , Neoplasias/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Galectina 1/farmacologia , Galectinas/farmacologia , Humanos , Imunoprecipitação , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Neoplasias/patologia , Neuraminidase/metabolismo , Oligossacarídeos/metabolismo , Ligação Proteica , Receptores de Fibronectina/metabolismo
14.
Prog. obstet. ginecol. (Ed. impr.) ; 53(7): 261-266, jul. 2010. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-80643

RESUMO

Objetivo. Comparar dos formas de abordar el trabajo prematuro de parto. La primera basándonos exclusivamente en criterios clínicos y la segunda empleando como herramientas auxiliares la prueba de la fibronectina y la longitud cervical por ecografía vaginal. Material y métodos. Estudio comparativo de ambas estrategias, enfatizando en costes hospitalarios y resultados perinatales. Para el grupo de estudio, en el que empleábamos ambos marcadores para seleccionar a las mujeres de mayor riesgo, empleamos un grupo prospectivo de 122 gestantes que acudieron de urgencia por amenaza de parto pretérmino (APP), y el grupo control (n=112) formado con una cohorte histórica de gestantes ingresadas por APP. Las gestantes catalogadas como de riesgo bajo para tener un parto prematuro eran dadas de alta en urgencias y controladas de forma ambulatoria. Se estimaron los valores predictivos de ambas pruebas y los resultados medidos fueron la tasa de prematuridad, las complicaciones neonatales, los días de hospitalización y los costes hospitalarios resultantes de la hospitalización, la medicación y las visitas posteriores. Resultados. Los resultados perinatales y las tasas de prematuridad en ambos grupos eran comparables. El uso de los tocolíticos y corticoides se redujo al emplear ambos marcadores. La estancia hospitalaria mediana fue 0 en el grupo de estudio (2,6 días cuando eran hospitalizadas), frente a 5 días en el grupo control. Los costes hospitalarios por paciente fueron de 446.24 € en el grupo de estudio (rango intercuartílico (IQ) 1.390,08) y de 1.634,04 € (IQ 1.092,65) en el grupo control. Conclusiones. Empleando estas técnicas para el diagnóstico del verdadero trabajo prematuro de parto, y obteniendo resultados perinatales comparables, no está justificado el tratamiento universal de aquellas gestantes que consultan de urgencia por APP. Esta estrategia puede conducirnos a un ahorro aproximado de 1.200 € por gestante (AU)


Objective. To compare two strategies for the management of threatened preterm labor (TPL). The first strategy was based on clinical criteria alone, while the second used rapid fibronectin testing and cervical length measured by vaginal ultrasound. Material and methods. We compared the costs and perinatal outcomes of both strategies. In the study group, both markers were used to select women at highest risk. The study group consisted of a prospective group of 122 women attending the emergency department for TPL. The control group (n=112) was composed of a historical cohort of women admitted for TPL. Pregnant women classified as low risk for premature birth were discharged from the emergency department and were monitored on an outpatient basis. The sensitivity and specificity of both tests in predicting preterm labor were estimated. The results measured were prematurity < 37 weeks, neonatal complications, length of hospital stay and costs resulting from admission, medication and subsequent follow-up visits. Results. Prematurity and perinatal outcomes were similar in both groups. The use of tocolytics and corticosteroids was reduced by employing the two markers. The median length of hospital stay was 0 days in the study group (2.6 days among hospitalized patients) and 5 days in the control group. The costs incurred per patient were 446.24 euros in the study group (IQR: 1,390.08) and 1,634.04 euros (IQR: 1,092.65) in the control group. Conclusions. Based on the use of these techniques to select patients with true preterm labor and the similar perinatal results obtained in both groups, we conclude that universal treatment of all women with suspected preterm labor is not warranted. This strategy saves approximately 1,200 € per patient (AU)


Assuntos
Humanos , Feminino , Gravidez , Adulto , Trabalho de Parto Prematuro/diagnóstico , Trabalho de Parto Prematuro/epidemiologia , Receptores de Fibronectina/análise , Custos e Análise de Custo/métodos , /tendências , /economia , Trabalho de Parto Prematuro/economia , Estudos Prospectivos , Triagem Neonatal/tendências , Triagem Neonatal , Valor Preditivo dos Testes , Idade Gestacional , Estudos de Coortes , Sensibilidade e Especificidade , Trabalho de Parto/fisiologia
15.
Development ; 137(14): 2439-49, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20570943

RESUMO

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the alpha5 and alphav integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin alpha5-floxed mouse line and ablated alpha5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin alpha5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin alpha5 and alphav and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both alpha5 and alphav integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial alpha5 and alphav integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin alpha5 knockout endothelial cells and markedly reduced in integrin alpha5/alphav double-knockout endothelial cell lines. Therefore, neither alpha5 nor alphav integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.


Assuntos
Integrina alfa5/metabolismo , Integrina alfa5/fisiologia , Integrina alfaV/metabolismo , Integrina alfaV/fisiologia , Integrinas/fisiologia , Animais , Vasos Sanguíneos/metabolismo , Adesão Celular , Diferenciação Celular , Linhagem Celular , Endotélio/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibronectinas/fisiologia , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III , Receptores de Fibronectina/metabolismo , Receptores de Fibronectina/fisiologia
16.
Mol Biol Cell ; 21(14): 2514-28, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20505078

RESUMO

Exposure of cells to certain cytokines can alter how these same cells respond to later cues from other agents, such as extracellular matrix or growth factors. Interferon (IFN)-gamma pre-exposure inhibits the spreading of fibroblasts on fibronectin. Expression of the IFN-gamma-induced GTPase murine guanylate-binding protein-2 (mGBP-2) can phenocopy this inhibition and small interfering RNA knockdown of mGBP-2 prevents IFN-gamma-mediated inhibition of cell spreading. Either IFN-gamma treatment or mGBP-2 expression inhibits Rac activation during cell spreading. Rac is required for cell spreading. mGBP-2 also inhibits the activation of Akt during cell spreading on fibronectin. mGBP-2 is incorporated into a protein complex containing the catalytic subunit of phosphatidylinositol 3-kinase (PI3-K), p110. The association of mGBP-2 with p110 seems important for the inhibition of cell spreading because S52N mGBP-2, which does not incorporate into the protein complex with p110, is unable to inhibit cell spreading. PI3-K activation during cell spreading on fibronectin was inhibited in the presence of mGBP-2. Both IFN-gamma and mGBP-2 also inhibit cell spreading initiated by platelet-derived growth factor treatment, which is also accompanied by inhibition of Rac activation by mGBP-2. This is the first report of a novel mechanism by which IFN-gamma can alter how cells respond to subsequent extracellular signals, by the induction of mGBP-2.


Assuntos
Movimento Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Interferon gama/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas rac de Ligação ao GTP/metabolismo , Substituição de Aminoácidos/genética , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Integrina alfa4/metabolismo , Melanoma/patologia , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Fibronectina/metabolismo
17.
Immunology ; 129(2): 248-56, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19824923

RESUMO

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.


Assuntos
Movimento Celular/imunologia , Malária/imunologia , Plasmodium berghei/imunologia , Células Precursoras de Linfócitos T/metabolismo , Timo/metabolismo , Animais , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Células Cultivadas , Quimiocinas/biossíntese , Quimiocinas/genética , Quimiocinas/imunologia , Regulação da Expressão Gênica , Malária/parasitologia , Malária/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Plasmodium berghei/patogenicidade , Células Precursoras de Linfócitos T/imunologia , Células Precursoras de Linfócitos T/parasitologia , Células Precursoras de Linfócitos T/patologia , Receptores de Citoadesina/biossíntese , Receptores de Citoadesina/genética , Receptores de Citoadesina/imunologia , Receptores de Fibronectina/biossíntese , Receptores de Fibronectina/genética , Receptores de Fibronectina/imunologia , Receptores de Laminina/biossíntese , Receptores de Laminina/genética , Receptores de Laminina/imunologia , Timo/imunologia , Timo/parasitologia , Timo/patologia
18.
Cell Signal ; 22(3): 427-36, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19892014

RESUMO

Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.


Assuntos
Integrina beta1/metabolismo , Mastócitos/enzimologia , Proteínas Proto-Oncogênicas c-fes/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Fibronectina/metabolismo , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Integrina alfa5beta1/metabolismo , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fes/deficiência , Proteínas Proto-Oncogênicas c-fes/genética , Fator de Células-Tronco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Domínios de Homologia de src
19.
IUBMB Life ; 61(7): 731-8, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19514020

RESUMO

The formation, maturation, and dissolution of focal adhesions are basic prerequisites of cell migration and rely on the recruitment, signalling, and endocytosis of integrins. In many instances, extracellular matrix molecules are recognised by a number of integrins, and it is the sequential involvement of different integrins that allows establishment of cell polarity and migration towards a matrix stimulus. In this review, we consider both the similarities and differences between two key fibronectin receptors, alpha(v)beta(3) and alpha(5)beta(1) integrin. By considering the GTPase and kinase signalling and trafficking of two such closely-related receptors, we begin to understand how cell migration is coordinated.


Assuntos
Integrina alfa5beta1/fisiologia , Integrina alfaVbeta3/fisiologia , Receptores de Fibronectina/fisiologia , Transdução de Sinais/fisiologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Microdomínios da Membrana/fisiologia , Transporte Proteico , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
Curr Pharm Des ; 15(12): 1309-17, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19355970

RESUMO

Extracellular matrix (ECM) is composed of large collagen fibrils. Glycoproteins, such as fibronectin, can bind to collagen or form their own networks. Collagen fibrils are also decorated by proteoglycans, proteins that have large glycosaminoglycan sidechains. In addition, extracellular space often contains hyaluronan, a large glycosaminoglycan molecule that has no core protein. Basement membranes represent a specialized form of extracellular matrix. Basement membranes are built by laminin and type IV collagen networks. In multicellular animals cells are anchored to ECM and basement membranes. Cell locomotion during development and after tissue injury is also based on cellular interactions with different matrix molecules. Specific cell surface receptors mediate these interactions. The largest family of receptors, which mediates cell adhesion to fibronectin, laminins and collagens is called the integrins. Several other cellular receptors have also evolved to bind to various matrix components. Here, we review the basic facts about these receptors and shortly describe their role in human diseases, including cancer and inflammation.


Assuntos
Matriz Extracelular/fisiologia , Animais , Matriz Extracelular/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Receptores de Colágeno/metabolismo , Receptores de Fibronectina/metabolismo , Receptores de Laminina/metabolismo
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