RESUMO
GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Adesão Celular , Tamanho Celular , Colágeno/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Receptores de Hialuronatos/metabolismo , Laminina/metabolismo , Hipófise/citologia , Hipófise/efeitos dos fármacos , Ratos , Receptores de Fibronectina/metabolismo , Receptores de Laminina/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Células Tumorais CultivadasRESUMO
Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.
Assuntos
Glicosaminoglicanos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Fibronectina/metabolismo , Adesão Celular , Eletroforese em Gel Bidimensional , Heparitina Sulfato/metabolismo , Humanos , Conformação Proteica , Sulfatos/metabolismo , Células Tumorais CultivadasRESUMO
Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha 5 beta 1 integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation and a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.
Assuntos
Fibronectinas/fisiologia , Receptores de Fibronectina/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrina/metabolismo , Processamento de Proteína Pós-Traducional , Sulfatos/metabolismoRESUMO
We have previously reported that CD7 expressed on resting human NK cells is a signal-transducing molecule, which upon ligation with mAb induces a rapid increase in cytoplasmic free calcium, secretion of IFN-gamma, and augmented NK activity against K562 targets. We now demonstrate that Ab-mediated clustering of CD7 molecules on NK cells results in enhanced phosphorylation on tyrosine residues of intracellular proteins of 60, 70, 80, 97, and 120 kDa. In the presence of genistein, a specific inhibitor of protein tyrosine kinase, the enhanced level of tyrosine phosphorylation was blocked, indicating that CD7 may induce signaling via activation of tyrosine kinases. Cross-linking of CD7 or CD16 molecules with primary and secondary Abs, as well as stimulation of NK cells with phorbol ester (PMA) or with calcium ionophore A23187 also induced beta 1 integrin-mediated adhesion of these cells to fibronectin (FN)-coated plastic surfaces. In contrast, cross-linking of CD2 expressed on the surface of NK cells had no significant effect on NK cell adhesion to FN. This adhesion was not associated with up-regulation of expression of alpha 4 beta 1 or alpha 5 beta 1 FN receptors on NK cells, but it required an intact cytoskeleton. The CD7-induced adhesion to FN was mediated by alpha 4 beta 1 and alpha 5 beta 1 integrins, as it was partially blocked by FN connective segment-1 peptide (EILDVPST), the alpha 4 beta 1-binding domain, as well as by RGD-containing peptides, the alpha 5 beta 1-binding domain, but not by EILEVPST or RGE control peptides. NK cell binding to FN was also partially inhibited by mAb to alpha 4, alpha 5, and beta 1 integrins. The mechanism by which cross-linking of CD7 or CD16 on NK cells induced adhesion to FN appeared to involve both protein tyrosine kinase and protein kinase C, because this adhesion was blocked in the presence of either genistein or a protein kinase C inhibitor, staurosporin. Our data demonstrate that signals transduced via triggering of either CD7 or CD16 molecules are involved in the regulation of the functional activity of beta 1 integrins on NK cells.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Fibronectinas/metabolismo , Integrinas/metabolismo , Células Matadoras Naturais/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Fibronectina/metabolismo , Receptores Imunológicos/fisiologia , Tirosina/análogos & derivados , Alcaloides/farmacologia , Sequência de Aminoácidos , Antígenos CD7 , Cálcio/fisiologia , Adesão Celular/efeitos dos fármacos , Citocalasina B/farmacologia , Ativação Enzimática , Genisteína , Humanos , Técnicas In Vitro , Isoflavonas/farmacologia , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Fosfotirosina , Receptores de IgG/fisiologia , Transdução de Sinais , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Tirosina/metabolismoRESUMO
Extracellular matrix (ECM) proteins can influence cell migration and differentiation in a variety of cell systems. Within the thymus, these molecules are heterogeneously distributed, and their physiological role is poorly understood. This prompted us to carry out in vitro studies using the thymic nurse cell (TNC) model. We observed that fibronectin and laminin accelerate spontaneous in vitro release of thymocytes from TNC, whereas anti-ECM antibodies exhibited a blocking effect. Similar results were obtained with anti-ECM receptor reagents. Moreover, these antibodies abrogated in vitro reconstitution of TNC complexes and thymocyte adhesion to TNC-derived epithelial cultures. Our results indicate that lymphocyte traffic in TNC (comprising both entrance into and exit from the epithelial structure) is affected by interactions involving extracellular matrix ligands and receptors. In this respect, the dynamic analysis of thymic nurse cell complexes should be regarded as a relevant in vitro tool for functional studies of distinct adhesion molecules in intrathymic lymphocyte traffic.
Assuntos
Moléculas de Adesão Celular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Timo/citologia , Animais , Proteínas de Transporte/metabolismo , Adesão Celular , Células Cultivadas , Feminino , Receptores de Hialuronatos , Imuno-Histoquímica , Integrinas/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos , Receptores de Superfície Celular/metabolismo , Receptores de Fibronectina/metabolismo , Receptores de Retorno de Linfócitos/metabolismoRESUMO
Aberrant glycosylation is a common feature of neoplastic cells. Although described for many years, the role of aberrant patterns of glycosylation is not fully understood. Our group has been focusing on the role of glycosylation in cell:matrix interactions, such as adhesion, spreading and migration on defined substrata (e.g., laminin and fibronectin). Animal lectins, such as galaptins, also seem to be involved in these processes.