RESUMO
We previously reported that H-ras-induced metastatic ability in murine NIH 3T3 cells is accompanied by increased expression of osteopontin (OPN). OPN is a secreted phosphoprotein that contains a GRGDS amino acid sequence, suggesting adhesive function, but the function of OPN in tumor cells remains poorly understood. Here we report that PAP2 cells (ras-transformed, metastatic NIH 3T3 cells) adhere and spread on OPN-coated substrates, while NIH 3T3 cells adhere and spread poorly on OPN. A similar pattern was seen for adhesion to laminin, while both cell lines adhered equally well to fibronectin. Adhesive interactions to OPN, laminin, and fibronectin were specific and were blocked by GRGDS (but not control GRGESP) peptides. The kinetics of adhesion to all three substrates was examined. Maximum adhesion was observed at 30-60 min, with reduced adhesion thereafter. We also purified metabolically labeled [32P]OPN secreted by PAP2 cells. Labeled OPN bound better in solution to PAP2 cells than to NIH 3T3 cells, and binding to both cell lines was blocked by GRGDS peptides, results that are consistent with the adhesion and spreading of these cells to OPN-coated substrates. Malignant PAP2 cells thus not only secrete increased levels of OPN, relative to NIH 3T3 cells, but also adhere better to this protein. While the target of OPN secreted by tumor cells is not known, our results raise the possibility that tumor cells that secrete OPN may also bind this protein and that this binding may function in autocrine-type signal transduction important to malignancy.
Assuntos
Células 3T3/patologia , Transformação Celular Neoplásica/patologia , Fibronectinas/fisiologia , Genes ras/fisiologia , Laminina/fisiologia , Sialoglicoproteínas/fisiologia , Células 3T3/metabolismo , Células 3T3/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Transformação Celular Neoplásica/genética , Fibronectinas/metabolismo , Laminina/metabolismo , Camundongos , Dados de Sequência Molecular , Metástase Neoplásica , Oligopeptídeos/metabolismo , Osteopontina , Radioisótopos de Fósforo , Ligação Proteica , Sensibilidade e Especificidade , Sialoglicoproteínas/metabolismoRESUMO
Osteopontin (OPN) has been associated with enhanced malignancy in breast cancer, but its functional role in this disease is poorly understood. To study the effect of OPN on cellular invasiveness, basal OPN expression was first assessed in members of a progression series of human mammary epithelial cell lines (21PT: immortalized, non-tumorigenic; 21NT: weakly tumorigenic; 21MT-1: tumorigenic, weakly metastatic; MDA-MB-435 cells: tumorigenic, highly metastatic). The two lines which expressed lowest basal levels of OPN (21PT, 21NT) were then examined for up-regulation of invasive behavior in response to exogenous or transfected (endogenous) OPN. Both 21PT and 21NT showed increased invasiveness through Matrigel when human recombinant (hr)OPN was added to the lower chamber of transwells. Both also showed a cell migration response to hrOPN. Populations of 21PT and 21NT cells stably transfected with an OPN-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness. Treatment of the parental 21PT and 21NT cells with exogenous hrOPN resulted in increased uPA mRNA expression and increased urokinase activity of the conditioned media. Both increased cell migration and induction of uPA expression are thus potential mechanisms of increased invasiness of breast epithelial cells in response to OPN.
Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Proteínas de Neoplasias/metabolismo , Sialoglicoproteínas/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Transformada , Colágeno , Meios de Cultivo Condicionados , Progressão da Doença , Combinação de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Matriz Extracelular , Feminino , Humanos , Laminina , Osteopontina , Proteoglicanas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/farmacologia , Transfecção , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
We have recently shown that either exogenous or endogenous, transfected OPN induces both uPA expression and increased invasiveness of 21 PT (non-tumorigenic) and 21 NT (tumorigenic) human mammary epithelial cells. Here we asked whether uPA contributes functionally to the increased invasiveness of these cells. The most invasive OPN-transfected cells were assessed for migration through Matrigel in transwell assays, in the presence or absence of various blocking antibodies and uPA inhibitors. Antibodies to both uPA and uPA receptor (uPAR) were shown to significantly inhibit cell invasion, as did the uPA inhibitors (plasminogen activator inhibitor-1 [PAI-1], p-aminobenzamidine [PABN], aprotinin, and amiloride). Both anti-uPA and anti-uPAR antibodies inhibited invasion to a level comparable to that of the control vector transfected cells. In contrast, non-specific IgG showed no antiinvasive effect. Cell migration experiments performed with the parental cell lines in the presence or absence of anti-uPA or anti-uPAR antibodies showed that uPA is also required for migratory responsiveness to exogenous OPN. These data thus provide direct evidence that OPN-induced invasion and migration of these cells requires uPA.
Assuntos
Mama/citologia , Movimento Celular/efeitos dos fármacos , Células Epiteliais/citologia , Sialoglicoproteínas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Northern Blotting , Adesão Celular , Colágeno , Combinação de Medicamentos , Células Epiteliais/metabolismo , Feminino , Humanos , Laminina , Osteopontina , Proteoglicanas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Células Tumorais CultivadasRESUMO
Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione-S-transferase (GST). We report that the GST-OPN fusion protein has functional activity. PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin-mediated mechanisms. Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein. Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein. Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post-translational modifications that occur in native OPN for adhesive function as measured here.
Assuntos
Glutationa Transferase/farmacologia , Oligopeptídeos/antagonistas & inibidores , Fosfoproteínas/farmacologia , Sialoglicoproteínas/farmacologia , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Transformada , DNA Complementar/genética , Escherichia coli , Humanos , Camundongos , Dados de Sequência Molecular , Osteopontina , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais CultivadasRESUMO
Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN-induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non-tumorigenic; 21NT, tumorigenic; non-metastatic; and MDA-MB-435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN-induced migration of 21PT and 21NT cells was alphavbeta5 and beta1-integrin dependent, and alphavbeta3-independent, while that of MDA-MB-435 cells was alphavbeta3-dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN-induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met.
Assuntos
Mama/citologia , Movimento Celular/efeitos dos fármacos , Células Epiteliais/fisiologia , Integrinas/metabolismo , Fosfoproteínas/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Sialoglicoproteínas/farmacologia , Northern Blotting , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Osteopontina , Fosfotirosina/metabolismo , Testes de Precipitina , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologiaRESUMO
Osteopontin (OPN), a secreted phosphoprotein, has been implicated in various biological phenomena (e.g. bone development, sepsis, tumor progression, and metastasis). Its role in any context is poorly understood. OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and binds to cells via integrin-mediated mechanisms. Using recombinant human osteopontin-glutathione S-transferase fusion protein and our improved hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal antibodies against osteopontin. We characterized two antibodies that recognize not only recombinant but also native human osteopontin. These antibodies do not cross-react with mouse osteopontin (recombinant protein or that secreted by ras-transformed NIH 3T3 cells), or bovine bone osteopontin, suggesting that they recognize epitopes unique to human OPN. One antibody specifically inhibited adhesion of MDA-MB-435 human breast cancer cells and ras-transformed NIH 3T3 cells to human osteopontin. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showed that thrombin cleavage reduces osteopontin cell binding activity. Thus we postulate that this monoclonal antibody recognizes and interferes with the function of the RGD/thrombin cleavage region of human OPN.
Assuntos
Anticorpos Monoclonais/imunologia , Adesão Celular , Oligopeptídeos/farmacologia , Sialoglicoproteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Bovinos , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/antagonistas & inibidores , Osteopontina , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Sialoglicoproteínas/imunologiaRESUMO
Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins ("delRGD" or "RGE") supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.