RESUMEN
The receptors for urokinase plasminogen activator were studied in both normal human fibroblasts (WI-38 cells) and their SV40-transformed counterpart (VA-13 cells). We have shown that transformed cells expose 10 times more urokinase plasminogen activator receptors (u-PAR) than normal cells. By cross-linking aliquots of cell lysates with the aminoterminal fragment of the A chain of u-PA, containing the receptor-binding sequence, we have observed a u-PAR concentration at focal contacts in both cell lines. Only transformed cells were able to efficiently invade the basement membrane Matrigel. Switching off the receptor gene expression by the anti-messenger oligodeoxynucleotides strategy abolished the invasive properties of transformed cells. The anti-messenger oligodeoxynucleotide sequence we have designed inhibited the u-PAR gene expression, lowering both the receptor and the receptor mRNA. This indicates that overexpression of u-PAR gene is itself responsible for invasivity of transformed fibroblasts in our cell model system and that antisense compound therapy may prove to be of clinical interest in the control of cancer spreading.
Asunto(s)
Invasividad Neoplásica , Oligonucleótidos Antisentido/farmacología , Receptores de Superficie Celular/metabolismo , Secuencia de Bases , Transformación Celular Viral , Células Cultivadas , Fibroblastos , Humanos , Datos de Secuencia Molecular , Activadores Plasminogénicos/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Virus 40 de los Simios , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Evidence indicates that breakdown of articular cartilage resulting in the loss of normal joint function is the distinctive feature of osteoarthritis. Degradation of cartilage extracellular matrix components involves the action of at least 2 classes of proteinases: serine proteinases and metalloproteinases. Receptors have been described on a wide range of cell lines for many such proteinases [urokinase-type plasminogen activator (u-PA), plasminogen/plasmin, collagenase], which subsequently activate each other on the solid phase of the cell surface, leading to cartilage destruction. We review the leading role of u-PA and its receptor (u-PAR) in cartilage degradation.
Asunto(s)
Cartílago Articular/fisiopatología , Osteoartritis/fisiopatología , Receptores de Superficie Celular/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Cartílago Articular/enzimología , Humanos , Técnicas In Vitro , Osteoartritis/enzimología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.
Asunto(s)
Fibroblastos/metabolismo , Receptores de Superficie Celular/metabolismo , Sistemas de Mensajero Secundario , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Células Clonales , Regulación hacia Abajo , Fibroblastos/citología , Humanos , Ratones , Mitógenos/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Acetato de Tetradecanoilforbol/farmacología , Transducción GenéticaRESUMEN
We studied non-mitogenic signal transduction in a human cell line of epidermal origin which is induced to chemotaxis following stimulation with human urokinase-type plasminogen activator (u-PA) or with the amino-terminal fragment (ATF) of u-PA A chain, which specifically interacts with the cellular receptor. U-PA and ATF stimulated the formation of diacylglycerol (DAG) independently of inositol lipid and phosphatidylcholine turnover, but concomitantly with de novo synthesis from glucose, thus resembling the DAG neosynthesis activated by insulin. DAG was measured in normal epidermal cells and in cells transfected with the human u-PA receptor (u-PAR) gene and stimulated with u-PA or ATF. Transfected clones showed an increase of cell motility under an ATF gradient in vitro as well as an increase of DAG production. These findings identify a novel mechanism of second messenger formation that conveys chemotactic signals upon stimulation of the u-PAR.
Asunto(s)
Epidermis/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Línea Celular , Quimiotaxis , Diglicéridos/biosíntesis , Glucosa/metabolismo , Glicerol/metabolismo , Humanos , Fragmentos de Péptidos/farmacología , Fosfatidilcolinas/metabolismo , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , TransfecciónRESUMEN
OBJECTIVE: Since the plasminogen activator [PA/plasminogen activator inhibitor (PAI) system is believed to be involved in a breakdown of articular cartilage in osteoarthritis (OA), we studied the modulation of single components of the fibrinolytic system (urokinase-type plasminogen activator, u-PA; plasminogen activator inhibitor-1, PAI-1; the surface receptor for u-PA, u-PAR) in human chondrocytes in the presence of piroxicam. METHODS: The drug was added to the chondrocyte culture medium either directly or by supplementing chondrocyte cultures with synovial fluid (SF) from patients with OA treated with piroxicam. We have shown u-PAR M(r) 55000 Da on human chondrocytes in suspension culture by cross linking chondrocyte lysates with 125I-labelled amino-terminal fragment (ATF) of human u-PA, which frames the sequence that specifically interacts with u-PAR. RESULTS: Such receptors decrease upon incubation of chondrocytes with piroxicam or with SF from patients treated with piroxicam. The culture medium of treated chondrocytes showed decreased fibrinolytic activity when compared with untreated controls, while PAI activity was increased in both SF chondrocyte culture medium following piroxicam treatment. At the same time, internalization of u-PA/u-PAR complexes increased after incubation of chondrocytes with piroxicam or PAI-1 rich SF. CONCLUSION: Our results indicate that the drug induces the surface clearance u-PAR by internalization of u-PA/PAI-/u-PAR complexes. Thus piroxicam reduces both the soluble fibrinolytic activity of human chondrocytes (increase of PAI activity and decrease of released u-PA) and the cell associated u-PA activity (clearance of u-PAR by internalization). The drug dependent changes in the fibrinolytic system suggest that piroxicam may be useful in preventing or limiting perilacunar cartilage damage in OA.
Asunto(s)
Cartílago Articular/metabolismo , Fibrinólisis/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Piroxicam/farmacología , Autorradiografía , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Células Cultivadas , Humanos , Osteoartritis/metabolismo , Osteoartritis/patología , Piroxicam/análisis , Piroxicam/uso terapéutico , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Líquido Sinovial/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
We have shown the presence of surface receptors for the amino-terminal fragment (ATF) of human urokinase-type plasminogen activator (u-PA) on an in vitro-established cell line of human epidermal origin by both radio-binding assays with human 125I-u-PA-ATF and transmission electron microscopy of a gold-u-PA complex. On the basis of cross-linking experiments with 125I-u-PA-ATF and subsequent autoradiography of the gels we have observed that such receptors are not spontaneously released into the culture medium. The treatment with phosphatidylinositol-specific phospholipase C induces the release of the receptor, which behaves as a glycosyl phosphatidyl inositol(GPI)-anchored protein. Phase-partitioning experiments on cell lysates have shown that the receptor partitions into the detergent phase. By detaching cell monolayers with the chelating agent EDTA we have prepared the cell-substratum contact sites of these cells, which represent only the 3.5% of the surface membrane of monolayered cells. Such plasma membrane remnants are highly selected since they contain about 43% of total u-PA-ATF binding sites. Such binding sites show the same biochemical and morphological characteristics of u-PA-ATF receptors observed in the monolayered cells, thus indicating that u-PA is selectively concentrated at the level of cell-substratum contacts. This is likely to enable directional proteolysis for cell migration and invasion.
Asunto(s)
Adhesión Celular , Epidermis/química , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/análisis , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Sitios de Unión , Línea Celular , Membrana Celular/química , Membrana Celular/ultraestructura , Reactivos de Enlaces Cruzados/farmacología , Epidermis/ultraestructura , Humanos , Microscopía Electrónica , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/farmacología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Succinimidas/farmacologíaRESUMEN
The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.
Asunto(s)
Desoxiglucosa/metabolismo , Proteínas de Transporte de Monosacáridos/biosíntesis , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Membrana Celular/metabolismo , Citocalasina B/farmacología , Diglicéridos/biosíntesis , Fibroblastos , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Humanos , Cinética , Pulmón , Ratones , Fragmentos de Péptidos/metabolismo , Proteína Quinasa C/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/biosíntesis , Virus 40 de los Simios , Piel , Fracciones Subcelulares/enzimología , TransfecciónRESUMEN
The aims of this study were: (1) to test the accuracy of flow cytometery (FC) in the measurement of DNA content in human solid tumors, (2) to correlate the FC DNA-index (DI) with the chromosome modal number (CMN) provided by cytogenetic analysis (CG), and (3) to investigate the most frequent pitfalls in FC histograms classification. FC and CG analyses were performed in parallel on 113 samples of human solid tumors of different origin. FC provided an evaluable histogram in 110 out of 113 cases (97%), whereas a successful CG culture was obtained in 79 out of 113 samples (72%). In the 79 cases evaluable by both FC and CG, a concordant ploidy status was found in 66 cases (84%) (47 diploid and 19 aneuploid) (P < 0.001, chi-square test). In the 19 concordant aneuploid tumors a close correlation between the CMN and the DI was found (y = 0.019 x + 0.151; r = 0.860). Concerning the 13 discordant cases, 11 (85%) were classified as aneuploid by FC and as diploid by CG, while 2 cases (15%) were CG aneuploid (1 near-diploid and 1 tetraploid) and FC diploid. The current study suggests that FC is a reliable method for the measurement of tumor DNA content of the studied solid tumors. Special attention should be paid to the improvement of DNA histograms quality, in order to reduce the difficulties in the detection of near-diploid and near-tetraploid cell populations. Multiple sampling should be warranted whenever possible.