RESUMO
This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.
Assuntos
Astrocitoma/metabolismo , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Proto-Oncogênicas , Motivos de Aminoácidos , Animais , Ligação Competitiva , Western Blotting , Neoplasias Encefálicas/metabolismo , Células COS , Ciclo Celular , Divisão Celular , Nucléolo Celular/metabolismo , Células Cultivadas , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA Complementar/metabolismo , Deleção de Genes , Genes Dominantes , Vetores Genéticos/metabolismo , Glioma/metabolismo , Lectinas Tipo C , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , VersicanasRESUMO
Aggrecan is a member of the chondroitin sulphate (CS) proteoglycan family, which also includes versican/PG-M, neurocan and brevican. Members of this family exhibit structural similarity: a G1 domain at the N-terminus and a G3 domain at the C-terminus, with a central sequence for modification by CS chains. A unique feature of aggrecan is the insertion of three additional domains, an inter-globular domain (IGD), a G2 domain and a keratan sulphate (KS) domain (sequence modified by KS chains), between the G1 domain and the CS domain (sequence modified by CS chains). The G1 and G3 domains have been implicated in product secretion, but G2, although structurally similar to the tandem repeats of G1, performs an unknown function. To define the functions of each aggrecan domain in product processing, we cloned and expressed these domains in various combinations in COS-7 cells. The results indicated that the G3 domain enhanced product secretion, alone or in combination with the KS or CS domain, and promoted glycosaminoglycan (GAG) chain attachment. Constructs containing the G1 domain were not secreted. Addition of a CS domain sequence to G1 reduced this inhibition, but GAG chain attachment was still decreased. The potential GAG chain attachment site in the IGD was occupied by GAGs, and IGD product was secreted efficiently. The KS domain was modified by GAG chains and secreted. Finally, the G2 domain was expressed but not secreted, and inhibited secretion of the IGD when expressed as an IGD-G2 combination.
Assuntos
Proteínas da Matriz Extracelular , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Agrecanas , Animais , Sequência de Bases , Western Blotting , Células COS , Primers do DNA , Lectinas Tipo C , Proteoglicanas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This investigation was designed to characterize the effect of the extracellular matrix molecule versican on chondrocyte morphology, using the well-studied chondrocyte cell culture system. When cultured chondrocytes reverted or "dedifferentiated" to a fibroblast-like morphology, we found that versican expression was significantly enhanced. Transfection of chondrocytes, isolated from embryonic chicken sterna, with a chicken miniversican construct accelerated the reversion process, while expression of an antisense construct inhibited it. A mutant miniversican lacking two epidermal growth factor-like motifs (versicanDeltaEGF) promoted differentiation, as shown by morphological changes and changes in the expression of other extracellular matrix molecules. A truncated versican mutant, the G3DeltaEGF, a G3 domain lacking its two epidermal growth factor-like motifs, also enhanced differentiation. This effect is related to G3DeltaEGF-induced change in cytoskeleton, since transfected cells exhibited misassembly of actin filaments. This article thus provides the first evidence that versican modulates chondrocyte morphology via changes in cytoskeletal structure, and may imply that extracellular matrix molecules play an important role in cell differentiation.
Assuntos
Tamanho Celular , Condrócitos/citologia , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Fator de Crescimento Epidérmico/química , Actinas/metabolismo , Motivos de Aminoácidos , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Condrócitos/metabolismo , Proteoglicanas de Sulfatos de Condroitina/genética , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Vetores Genéticos , Humanos , Lectinas Tipo C , Morfogênese , Faloidina/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/genética , Proteoglicanas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , VersicanasRESUMO
The most abundant macromolecules in cartilage are hyaluronan, collagen, aggrecan, and link protein, which are believed to play roles in maintaining a unique three-dimensional network for a functional joint. This study was designed to investigate the roles of the major extracellular molecules in mediating chondrocyte-matrix interactions. We employed specific approaches to remove components individually or in combination: hyaluronan was digested with hyaluronidase; type II collagen was digested with collagenase; aggrecan expression was inhibited with antisense and beta-xyloside approaches; and link protein expression was inhibited with antisense oligonucleotides. Digestion of hyaluronan induced chondrocyte attachment to tissue culture plates, collagen-coated plates, and fibroblast-like chondrocyte cultures, and induced chondrocyte aggregation. Treated chondrocytes exhibited a fibroblast-like morphology, and the effects of hyaluronidase were dose-dependent. Conversely, the effect of collagenase on chondrocyte adhesion and aggregation was far less pronounced. Treatment with Arg-Gly-Asp peptide inhibited chondrocyte-collagen interaction. Chondrocyte attachment was enhanced by antisense oligonucleotides complementary to aggrecan and link protein and by beta-xyloside treatment. Nevertheless, hyaluronan seems to predominate over the other molecules in mediating chondrocyte-matrix interactions.
Assuntos
Adesão Celular/fisiologia , Agregação Celular/fisiologia , Movimento Celular/fisiologia , Condrócitos/citologia , Proteínas da Matriz Extracelular/fisiologia , Animais , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Hialuronoglucosaminidase/farmacologiaRESUMO
The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF- littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/fisiologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Subpopulações de Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fator Reumatoide/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Complexo Antígeno-Anticorpo/metabolismo , Subpopulações de Linfócitos B/metabolismo , Proteína Ligante Fas , Haptenos/imunologia , Teste de Histocompatibilidade , Temperatura Alta , Soros Imunes/farmacologia , Alótipos de Imunoglobulina/genética , Alótipos de Imunoglobulina/fisiologia , Imunoglobulina G/fisiologia , Ativação Linfocitária/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Camundongos Transgênicos , Nucleossomos/imunologia , Fator Reumatoide/biossíntese , Receptor fas/genéticaRESUMO
The large aggregating chondroitin sulfate proteoglycans, including aggrecan, versican (PG-M), neurocan, and brevican, are characterized by N-terminal and C-terminal globular (or selectin-like) domains known as the G1 and G3 domains, respectively. For this study, we generated a series of expression constructs containing various combinations of chicken versican/PG-M domains and a leading peptide of link protein in order to examine the roles of the G1 and G3 domains in versican function. In transfection studies, we observed that the presence of the G1 domain was sufficient to inhibit product secretion, while the G3 domain enhanced this process. We also demonstrated that the G1 domain inhibited the attachment of glycosaminoglycan chains to the core proteins, while the G3 domain enhanced this process. Further studies revealed that inhibition of secretion by G1 was mediated by its two tandem repeats, while G3's promotion of glycosaminoglycan chain attachment was apparently dependent on G3's complement-binding protein (CBP)-like motif. The modulatory effects of these two molecular domains may contribute to versican's biological activities.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/genética , Glicosaminoglicanos/metabolismo , Animais , Sítios de Ligação , Células COS , Galinhas , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas do Sistema Complemento/metabolismo , Lectinas Tipo C , Mutagênese Sítio-Dirigida , Proteoglicanas/química , Proteoglicanas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Mapeamento por Restrição , Transfecção , VersicanasRESUMO
Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.
Assuntos
Apoptose , Quinases relacionadas a CDC2 e CDC28 , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular , Fator de Crescimento Epidérmico/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma de Células Escamosas/patologia , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Colágeno/metabolismo , Ciclina A/biossíntese , Ciclina D1/biossíntese , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/biossíntese , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/metabolismo , Citometria de Fluxo , Humanos , Neoplasias Epiteliais e Glandulares/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteína do Retinoblastoma/biossíntese , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.
Assuntos
Apoptose/fisiologia , Condrócitos/metabolismo , Colágeno/metabolismo , Integrinas/metabolismo , Animais , Western Blotting , Agregação Celular , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Colagenases/metabolismo , Meios de Cultivo Condicionados , Matriz Extracelular/metabolismo , Citometria de Fluxo , Ácido Hialurônico/farmacologia , Hialuronoglucosaminidase/metabolismo , Ligação ProteicaRESUMO
Versican is a large extracellular proteoglycan and is expressed in a variety of tissues including the central nervous system. A malignant astrocytoma cell line U87 with high motility expressed a higher level of versican than another malignant astrocytoma cell line U343 with lower motility. We observed that the U87 cells were less adherent to tissue culture plates than the U343 cells. To investigate the role of versican in astrocytoma cell migration, we generated recombinant products of a mini-versican construct expressed in COS-7 cells. We found that the mini-versican products enhanced astrocytoma cell migration. Furthermore, enhanced migration was promoted by the G1 domain but not the G3 domain of versican. We introduced culture medium containing products of the mini-versican, the G1, and the G3 constructs separately into the astrocytoma cell lines U87 and U343. The mini-versican and the G1 construct, but not the G3 construct, were shown to reduce astrocytoma cell adhesion. The present data suggest that versican exerts its effect on astrocytoma cell migration and adhesion through the G1 domain.
Assuntos
Astrocitoma/patologia , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Estrutura Terciária de Proteína , Proteoglicanas/fisiologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Humanos , Lectinas Tipo C , Células Tumorais Cultivadas , VersicanasRESUMO
We have previously demonstrated that versican stimulated NIH3T3 fibroblast proliferation. Since versican is expressed in cartilage, we investigated whether versican plays a role in chondrocyte proliferation. We developed a technique to stably express a recombinant versican mini-gene in chicken chondrocytes, and its effect on chondrocyte proliferation was analyzed by the increase in cell number. The effect of cell adhesion on cell proliferation was tested. Finally, the versican mini-gene was truncated to assess the role of EGF-like motifs in cell proliferation. Expression of the recombinant versican mini-gene stimulated chondrocyte proliferation. Antisense oligonucleotides complementary to versican inhibited chondrocyte proliferation. The G1 domain of versican stimulated chondrocyte proliferation by destabilizing chondrocyte adhesion. Furthermore, deletion of the two EGF-like motifs from the G3 domain also reduced the function of versican in stimulating cell proliferation. Versican enhances chondrocyte proliferation through a mechanism involving its G1 and G3 domains. This finding may have implications for our understanding of the pathogenesis of various joint diseases.
Assuntos
Condrócitos/citologia , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Fragmentos de Peptídeos/fisiologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Animais , Comunicação Celular/genética , Comunicação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Proteoglicanas de Sulfatos de Condroitina/genética , Lectinas Tipo C , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/farmacologia , VersicanasRESUMO
Versican is a highly expressed proteoglycan in zones of developing tissues. To investigate whether versican plays a role in cell differentiation, we studied its role in mesenchymal condensation and chondrogenesis. Here we report that a mini-versican gene product inhibits mesenchymal chondrogenesis but not condensation. The mini-versican-treated mesenchymal cultures form fewer, smaller cartilaginous nodules and produced lower levels of link protein and type II collagen. The versican G3 domain alone, but not G1, was sufficient to inhibit mesenchymal chondrogenesis. Deletion of two epidermal growth factor (EGF)-like motifs in the G3 domain abolished the effect of versican. The G3 domain of aggrecan, which does not contain an EGF-like motif, did not inhibit mesenchymal chondrogenesis. We also generated a chimera construct containing the two EGF-like motifs of versican and the G3 domain of aggrecan, and we observed that this chimera construct inhibited chondrogenesis to a lesser extent than did the full-length versican G3 construct. Direct transfection of mesenchymal cells with different constructs produced similar results. Furthermore, treatment with versican antisense oligonucleotides and transfection with a versican antisense construct promoted chondrogenesis. Taken together, our results strongly suggest that versican inhibits mesenchymal chondrogenesis via its EGF-like motifs.
Assuntos
Condrogênese , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células 3T3 , Animais , Sequência de Bases , Células COS , Embrião de Galinha , Proteoglicanas de Sulfatos de Condroitina/química , Primers do DNA , Fator de Crescimento Epidérmico/química , Lectinas Tipo C , Mesoderma , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , VersicanasRESUMO
Numerous studies have shown that catecholamines can modulate lymphocyte migration. This effect may be mediated in part by modulation of lymphocyte-endothelial cell interactions, which is dependent on adhesion molecules expressed on both of these cells. Our results show that catecholamines decreased T-cell binding to IL-1 activated endothelial cells in vitro. The decrease in adhesion was not mediated by a change in adhesion molecule expression as LFA-1 and VLA-4 expression on T-cells and ICAM-1 and VCAM-1 expression on endothelial cells were not changed by catecholamine stimulation. T-cells flatten and enlarge the area of surface contact as they adhere to endothelial cells. Image analysis of the number of T-cells bound and the amount of cell spreading over several time points suggests that catecholamines alter the kinetics of T-cell-endothelial cell adhesion. These results support the hypothesis that catecholamines can alter lymphocyte-endothelial interactions in vivo, which in turn would affect lymphocyte migration.