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2.
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
3.
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
4.
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
5.
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
6.
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
7.
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 para 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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Arterioscler Thromb Vasc Biol ; 29(4): 571-8, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19122169

RESUMO

OBJECTIVE: Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. METHODS AND RESULTS: The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits alpha5 and beta1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. CONCLUSIONS: In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells.


Assuntos
Caspase 8/metabolismo , Células Endoteliais/enzimologia , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Células-Tronco/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome de Alstrom , Animais , Caspase 8/genética , Inibidores de Caspase , Adesão Celular , Movimento Celular , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Membro Posterior , Humanos , Integrina alfaV/metabolismo , Integrina beta1/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Camundongos , Camundongos Knockout , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Receptores de Fibronectina/metabolismo , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos
16.
BMC Dev Biol ; 9: 1, 2009 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-19126199

RESUMO

BACKGROUND: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development. RESULTS: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV). CONCLUSION: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.


Assuntos
Blastocisto/metabolismo , Fibronectinas/análise , Fibronectinas/fisiologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/fisiologia , Receptores de Fibronectina/análise , Receptores de Fibronectina/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Bovinos , Adesão Celular/genética , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Gravidez , Estrutura Terciária de Proteína/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Exp Eye Res ; 88(4): 689-93, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18835267

RESUMO

Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).


Assuntos
Fibronectinas/fisiologia , Malha Trabecular/fisiologia , Humor Aquoso/fisiologia , Fibronectinas/química , Humanos , Receptores de Fibronectina/fisiologia , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
18.
PLoS One ; 3(6): e2373, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19020673

RESUMO

Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10(th) type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal beta-strands - residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules - a method that propagates matrix assembly.


Assuntos
Fibronectinas/química , Membrana Celular/metabolismo , Cristalografia por Raios X , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Cinética , Modelos Biológicos , Oligopeptídeos/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Fibronectina/química , Solventes/química , Resistência à Tração
19.
Arterioscler Thromb Vasc Biol ; 28(10): 1703-13, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18658045

RESUMO

Angiogenesis, the formation of new blood vessels from preexisting vasculature, contributes to the pathogenesis of many disorders, including ischemic diseases and cancer. Integrins are cell adhesion molecules that are expressed on the surface of endothelial cells and pericytes, making them potential targets for antiangiogenic therapy. Here we review the contribution of endothelial and mural cell integrins to angiogenesis and highlight their potential as antiangiogenesis targets.


Assuntos
Endotélio Vascular/metabolismo , Integrinas/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Inibidores da Angiogênese/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/metabolismo , Receptores de Colágeno/metabolismo , Receptores de Fibronectina/metabolismo , Receptores de Laminina/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
J Cell Sci ; 121(Pt 14): 2360-71, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18577581

RESUMO

beta1 integrins are major cell surface receptors for fibronectin. Some integrins, including beta1 integrins, are known to undergo constitutive endocytosis and recycling. Integrin endocytosis/recycling has been implicated in the regulation of cell migration. However, the mechanisms by which integrin endocytosis/recycling regulates cell migration, and other biological consequences of integrin trafficking are not completely understood. We previously showed that turnover of extracellular matrix (ECM) fibronectin occurs via receptor-mediated endocytosis. Here, we investigate the biological relevance of beta1 integrin endocytosis to fibronectin matrix turnover. First, we demonstrate that beta1 integrins, including alpha5beta1 play an important role in endocytosis and turnover of matrix fibronectin. Second, we show that caveolin-1 constitutively regulates endocytosis of alpha5beta1 integrins, and that alpha5beta1 integrin endocytosis can occur in the absence of fibronectin and fibronectin matrix. We also show that downregulation of caveolin-1 expression by siRNA results in marked reduction of beta1 integrin and fibronectin endocytosis. Hence, caveolin-1-dependent beta1 integrin and fibronectin endocytosis plays a critical role in fibronectin matrix turnover, and may contribute to abnormal ECM remodeling that occurs in fibrotic disorders.


Assuntos
Caveolina 1/metabolismo , Endocitose , Fibronectinas/metabolismo , Integrina beta1/metabolismo , Animais , Clatrina/metabolismo , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Integrina alfa5/metabolismo , Camundongos , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de Fibronectina/metabolismo , Fatores de Tempo
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