RESUMEN
Integrin receptor activation is an important regulatory mechanism for cell-substrate and cell-cell adhesion. In this study, we explore a signaling pathway activated by mAb 12G10, an antibody that can activate beta(1) integrins and induce integrin-mediated cell-cell and cell-substrate adhesion. We have found that the cAMP-dependent protein kinase (PKA) is required for both mAb 12G10-induced cell-cell and cell-substrate adhesion of HT-1080 cells. Binding of mAb 12G10 to beta(1) integrins stimulates an increase in intracellular cAMP levels and PKA activity, and a concomitant shift in the localization of the PKA type II regulatory subunits from the cytoplasm to areas where integrins expressing the 12G10 epitope are located. MAb 12G10-induced cell-cell adhesion was mimicked by a combination of clustering beta(1) integrins and elevating PKA activity with Sp-adenosine-3',5'-cyclic monophosphorothioate or forskolin. We also show that two processes required for HT-1080 cell-cell adhesion, integrin clustering and F-actin polymerization are both dependent on PKA. Taken together, our data suggest that PKA plays a key role in the signaling pathway, resulting from activation of beta(1) integrins, and that this enzyme may be required for upregulation of cell-substrate and cell-cell adhesion.
Asunto(s)
Actinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Adhesión Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Integrina beta1/metabolismo , Actinas/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , AMP Cíclico/agonistas , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Citoplasma/metabolismo , Fibrosarcoma/metabolismo , Humanos , Integrina beta1/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.
Asunto(s)
Neoplasias de la Mama/patología , Colágeno/metabolismo , Ácidos Grasos Insaturados/farmacología , Integrina beta1/fisiología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Ácido Araquidónico/farmacología , Neoplasias de la Mama/enzimología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Grasas Insaturadas en la Dieta/farmacología , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Humanos , Immunoblotting , Integrina beta1/biosíntesis , Ácido Linoleico/farmacología , Ácido Oléico/farmacología , Proteína Quinasa C-epsilon , Estimulación Química , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.
Asunto(s)
Adhesión Celular/fisiología , Integrina beta1/metabolismo , Actinas/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cadherinas/metabolismo , Cationes Bivalentes/metabolismo , Colágeno/metabolismo , Relación Dosis-Respuesta Inmunológica , Epítopos , Fibronectinas/metabolismo , Fibrosarcoma , Humanos , Hibridomas , Inmunohistoquímica , Integrina beta1/inmunología , Laminina/metabolismo , Microscopía Confocal , Modelos Biológicos , Transducción de Señal , Tiazoles/farmacología , Tiazolidinas , Células Tumorales CultivadasRESUMEN
This unit describes the purification of the multifunctional adhesive glycoprotein fibronectin from plasma or of cell-derived fibronectin from cell surfaces and from conditioned medium. Fibronectin can be used in cell adhesion and migration assays, and can be obtained in relatively high purity using simple affinity chromatography techniques.
Asunto(s)
Medios de Cultivo Condicionados/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/sangre , Fibronectinas/aislamiento & purificación , Células Cultivadas , Técnicas Citológicas , HumanosRESUMEN
This unit describes the purification of extracellular vitronectin from plasma by a series of affinity chromatography steps. First plasma is depleted of fibronectin and other heparin-binding proteins; the sample is then treated with urea to activate the heparin-binding activity of vitronectin. The resulting vitronectin is approximately 98% pure.
Asunto(s)
Cromatografía de Afinidad/métodos , Matriz Extracelular/química , Vitronectina/sangre , Vitronectina/aislamiento & purificación , Animales , Humanos , Vitronectina/químicaRESUMEN
Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.
Asunto(s)
Ácido Araquidónico/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Colágeno/fisiología , Proteínas de Choque Térmico , Proteínas Serina-Treonina Quinasas/fisiología , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Proteínas de Choque Térmico HSP27 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Chaperonas Moleculares , Proteínas de Neoplasias/fisiología , Fosforilación , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Vinexin, a novel protein that plays a key role in cell spreading and cytoskeletal organization, contains three SH3 domains and binds to vinculin through its first and second SH3 domains. We show here that the third SH3 domain binds to Sos, a guanine nucleotide exchange factor for Ras and Rac, both in vitro and in vivo. Point mutations in the third SH3 domain abolished the vinexin-Sos interaction. Stimulation of NIH/3T3 cells with serum, epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) decreased the electrophoretic mobility of Sos and concomitantly inhibited formation of the vinexin-Sos complex. Phosphatase treatment of lysates restored the binding of Sos to vinexin, suggesting that signaling from serum, EGF, or PDGF regulates the vinexin-Sos complex through the Sos phosphorylation. To evaluate the function of vinexin downstream of growth factors, we examined the effects of wild-type and mutant vinexin expression on extracellular signal-regulated kinase (Erk) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation in response to EGF. Exogenous expression of vinexin beta in NIH/3T3 cells enhanced JNK/SAPK activation but did not affect Erk activation. Moreover mutations in the third SH3 domain abolished EGF activation of JNK/SAPK in a dominant-negative fashion, whereas they slightly stimulated Erk. Together these results suggest that vinexin can selectively modulate EGF-induced signal transduction pathways leading to JNK/SAPK kinase activation.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Musculares/metabolismo , Proteínas Son Of Sevenless/metabolismo , Células 3T3 , Animales , Cromatografía de Afinidad , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Ratones , Proteínas Musculares/química , Proteínas Musculares/aislamiento & purificación , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Mutación Puntual , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas Son Of Sevenless/química , Proteínas Son Of Sevenless/aislamiento & purificación , Transfección , Dominios Homologos srcRESUMEN
Invadopodia are membrane extensions of aggressive tumor cells that function in the activation of membrane-bound proteases occurring during tumor cell invasion. We explore a novel and provocative activity of integrins in docking proteases to sites of invasion, termed invadopodia. In the absence of collagen, alpha(3)beta(1) integrin and the gelatinolytic enzyme, seprase, exist as nonassociating membrane proteins. Type I collagen substratum induces the association of alpha(3)beta(1) integrin with seprase as a complex on invadopodia. The results show that alpha(3)beta(1) integrin is a docking protein for seprase to form functional invadopodia. In addition, alpha(5)beta(1) integrin may participate in the adhesion process necessary for invadopodial formation. Thus, alpha(3)beta(1) and alpha(5)beta(1) integrins play major organizational roles in the adhesion and formation of invadopodia, promoting invasive cell behavior.
Asunto(s)
Gelatinasas/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Serina Endopeptidasas , Animales , Antígenos de Superficie , Biomarcadores de Tumor/metabolismo , Colágeno/metabolismo , Endopeptidasas , Técnica del Anticuerpo Fluorescente , Integrina alfa3beta1 , Melanoma/metabolismo , Microscopía Fluorescente , Pruebas de Precipitina , Células Tumorales CultivadasRESUMEN
Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.
Asunto(s)
Toxinas Botulínicas , Integrinas/biosíntesis , Proteína Quinasa C/fisiología , ADP Ribosa Transferasas/metabolismo , ADP Ribosa Transferasas/farmacología , Animales , Neoplasias de la Mama , Adhesión Celular/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Integrinas/genética , Ratones , Naftalenos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Receptores de Colágeno , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Citoesqueleto/fisiología , Proteínas Musculares/genética , Vinculina/metabolismo , Dominios Homologos src , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Movimiento Celular , Pollos , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Conejos , Homología de Secuencia de Aminoácido , Porcinos , Distribución Tisular , Vinculina/genéticaRESUMEN
Endothelial cell function is regulated by interactions among cells, the extracellular matrix (ECM), and soluble mediators. We investigated this interaction by examining the effect of 17beta-estradiol (E2) on release of basic fibroblast growth factor (FGF-2) by human coronary artery endothelial cells (HCAEC) cultured on ECM proteins. After estrogen-depleted HCAEC were treated with E2 for 2 h, the conditioned media and cell layers were evaluated by immunoblot or ELISA for FGF-2. Release of FGF-2 into conditioned media was enhanced 10-fold compared to that on plastic and a further 2.4-fold by E2. As FGF-2 release from cells into the media increases, there is a corresponding decrease in the cellular content of FGF-2. By ELISA, FGF-2 release increased 406, 179, and 262%, on type IV collagen, laminin, or fibronectin, respectively. HCAEC cultured on type I collagen did not show E2-enhanced FGF-2 release by ELISA or immunoblot analysis. No changes were noted in HCAEC release of lactate dehydrogenase, tested as a control protein for cellular integrity. The estrogen receptor antagonist ICI182,780 blocked E2-induced, but not basal, FGF-2 release. Increased FGF-2 release occurred via a cycloheximide-insensitive pathway. Neither brefeldin-A nor genistein inhibited E2 enhancement of FGF-2 release by HCAEC cultured on fibronectin. However, the protein kinase C inhibitor calphostin C inhibited the E2-augmented FGF-2 release. These data show that E2 enhances FGF-2 release by HCAEC cultured on basement membrane proteins in the absence of wounding. This action requires the estrogen receptor and PKC activity, but does not require new protein synthesis, endoplasmic reticulum-to-Golgi-mediated secretion, or protein tyrosine phosphorylation. E2-enhanced FGF-2 release could contribute to the cardioprotective effects of estrogen.
Asunto(s)
Endotelio Vascular/metabolismo , Estradiol/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Brefeldino A/farmacología , Línea Celular , Vasos Coronarios/citología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Naftalenos/farmacología , Biosíntesis de ProteínasRESUMEN
The ninth type III module of murine fibronectin was expressed in E. coli and folded into a compact homogeneous monomer whose unfolding and refolding were then investigated by fluorescence, circular dichroism, calorimetry and electron microscopy. The isolated module is unusually labile under physiological conditions. When heated at 1 deg. C/minute it exhibits an irreversible endothermic transition between 35 and 42 degrees C depending on the protein concentration. The transition is accompanied by changes in secondary and tertiary structure with partial exposure of the single tryptophan and increased binding of the hydrophobic probe, 1,8-anilinonaphthalene-sulfonate. The partially unfolded intermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain substantial amounts of beta sheet structure. The multimers melt and dissociate reversibly in a second endothermic transition between 60 and 90 degrees C also depending on the protein concentration. This second transition destroys the remaining secondary structure and further exposes the tryptophan. Visualization of negatively stained specimens in the electron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of approximately 10 nm width and indeterminate length. A subdomain swapping mechanism is proposed in which beta strands from one partially unfolded molecule interact with complementary regions of another to form oligomers and polymers. The possibility that similar interactions could play a role in the formation of fibrils by fibronectin in vivo is discussed.
Asunto(s)
Amiloide/química , Fibronectinas/química , Animales , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cartilla de ADN , Ratones , Microscopía Electrónica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Termodinámica , Urea/químicaRESUMEN
We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.
Asunto(s)
Fibronectinas/química , Oligopéptidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Isótopos de Carbono , Fibronectinas/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Protones , Soluciones , Especificidad de la EspecieRESUMEN
The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.
Asunto(s)
Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Interleucina-7/farmacología , Linfocitos T/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Integrina beta1/metabolismo , Laminina/metabolismo , Ligandos , Linfocitos T/citología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Invasion of LOX human melanoma cells involves extracellular matrix (ECM) degradation and formation of cell surface invadopodia. Here we show that the ligation of alpha6beta1 by two peptides derived from the COOH-terminal globular domain of laminin-1 alpha1 chain (laminin G peptides), designated AG-10 (NPWHSIYITRFG) and AG-32 (TWYKIAFQRNRK), and antibodies against alpha6 and beta1 integrins promoted invasiveness. AG-10 and AG-32 inhibited cell adhesion on laminin, and the antibodies blocked cell adhesion on immobilized AG-10 and AG-32, suggesting that the peptides interact primarily with alpha6beta1 integrin. These soluble peptides and integrin antibodies induced invasiveness by causing an 2-3-fold increase in ECM degradation and invadopodial activity independently of adhesion activity of integrins that were prebound to ECM. The induced ECM degradation and invasion was associated with an increased surface expression of the 170-kDa membrane-bound gelatinase, seprase, as well as its intense localization at invadopodia but not at focal adhesions. However, the total expression levels of seprase, gelatinase A and beta1 integrins were not altered. We suggest that laminin G peptides act on the alpha6beta1 integrin signaling of invasion by stimulating invadopodial activities, which is distinct from their direct effects on cell adhesion on immobilized ECM.
Asunto(s)
Integrinas/fisiología , Laminina/farmacología , Melanoma/patología , Proteínas de la Membrana , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Serina Endopeptidasas , Secuencia de Aminoácidos , Anticuerpos/farmacología , Biomarcadores de Tumor , Adhesión Celular , Línea Celular , Endopeptidasas , Gelatinasas/biosíntesis , Humanos , Integrina alfa6beta1 , Integrinas/biosíntesis , Cinética , Laminina/metabolismo , Metaloproteinasa 2 de la Matriz , Melanoma/fisiopatología , Metaloendopeptidasas/biosíntesis , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Células Tumorales CultivadasRESUMEN
Cell adhesive interactions play important roles during many normal physiological processes such as embryonic development and wound repair, and also during the progression of diseases such as cancer. Cell adhesion is mediated by the specific interactions of cell surface receptors with extracellular glycoproteins. The best characterized cell adhesion receptors are the integrins. Integrins comprise a family of more than 23 noncovalent, heterodimeric complexes consisting of an alpha and a beta subunit. Each subunit is a glycoprotein with a large, globular extracellular domain and a transmembrane domain. Most integrins have relatively small cytoplasmic domains consisting of fewer than 60 amino acids. Although many integrins can bind fibronectin, the alpha 5, beta 1, integrin is the major fibronectin receptor on most cells. This integrin mediates such cellular responses to fibronectin substrates as adhesion, migration, assembly of extracellular matrix, and signal transduction. Integrin ligands, such as fibronectin, are not passive adhesive molecules but are active participants in the cell adhesive process that leads to signal transduction. The best characterized integrin ligand is fibronectin. Fibronectin is a multifunctional glycoprotein comprised of three different types of homologous repeating units (termed type I, type II, and type III). Fibronectin has at least two independent cell adhesive regions: one located near the center of the polypeptide chain in the ninth and tenth type III modules binds to the alpha 5 beta 1 integrin. The biological function of the central cell adhesive region requires two critical amino acid sequences--an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence, which function in synergy--for optimal binding to the alpha 5 beta 1 integrin. Furthermore, the spacing between the crucial RGD and PHSRN sequences is also important for activity, suggesting the sequences themselves are necessary, but not sufficient, to account for the cell adhesive activity of fibronectin. One of the manifestations of integrin-mediated signal transduction including protein tyrosine phosphorylation. One cytoplasmic protein that is phosphorylated in response to cell adhesion is the focal adhesion kinase known as pp125FAK or FAK. The beta 1, beta 3, and beta 5 integrin intracellular domains are sufficient to initiate signal transduction pathways. Furthermore, alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction. Other intracellular responses to cell adhesion include stimulation of migration, the assembly of an F-actin cytoskeleton and specialized structures called focal contacts, changes of cytoplasmic pH and calcium ion concentration, and modulation of proliferation and gene expression. Such varied modes of signal transduction are probably differentially controlled by a mechanism that requires either integrin receptor clustering alone, ligand occupancy in addition to clustering, or clustering and/or ligand occupancy plus tyrosine kinase activity for different responses. The examination of the fundamental mechanisms important for adhesion of cultured human cells and the resultant signaling processes has the potential of providing an understanding of molecular mechanisms involved in complex physiological processes and serving the basis for the development of novel therapeutic agents for the treatment of human disease.
Asunto(s)
Adhesión Celular/fisiología , Integrinas/fisiología , Transducción de Señal/fisiología , Fibronectinas/fisiología , HumanosRESUMEN
Integrin-ligand binding causes conformational changes in the integrin, as evidenced by the increased expression of epitopes known as ligand-induced binding sites. Some monoclonal antibodies (mAbs) that recognize ligand-induced binding sites stimulate ligand binding, possibly by stabilizing the ligand-occupied conformation of the integrin. Here we have investigated the effect of ligand recognition by alpha5beta1 on the binding of a mAb that inhibits beta1 integrin function (mAb 13). Ligand (fibronectin fragment or GRGDS peptide) decreased the binding of mAb 13 to alpha5beta1. Analysis of this inhibition showed that at high ligand concentrations, approximately 50% of the total integrin bound mAb 13 with >50-fold lower affinity than in the absence of ligand. The concentration of ligand required for half-maximal inhibition of antibody binding was independent of antibody concentration, suggesting that ligand acts as an allosteric inhibitor of mAb 13 binding. Hence, ligand and mAb 13 did not appear to compete directly for binding to alpha5beta1. The stimulatory anti-beta1 mAb 9EG7 was found to increase the maximum level of ligand binding approximately 2-fold, indicating that up to 50% of the total integrin could not bind ligand without 9EG7 stimulation. Analysis of mAb 13 binding in the presence of 9EG7 and ligand (i.e. maximal ligand occupancy) demonstrated that essentially all of the integrin bound mAb 13 with very low or zero affinity. Our results demonstrate that mAb 13 recognizes an epitope that is dramatically attenuated in the ligand-occupied form of alpha5beta1. Hence, since mAb 13 preferentially recognizes the unoccupied conformation of the integrin, the antibody may inhibit ligand binding by stabilizing the unoccupied state of alpha5beta1. In addition, we present evidence that the binding of mAb 13 to ligand-occupied alpha5beta1 may also induce a conformational change in the integrin, resulting in the displacement of ligand.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Integrina beta1/inmunología , Receptores de Fibronectina/química , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Unión Competitiva , Epítopos , Humanos , Ligandos , Datos de Secuencia Molecular , Oligopéptidos , Péptidos/química , Ratas , Receptores de Fibronectina/inmunologíaRESUMEN
Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Antígenos Bacterianos , Adhesión Bacteriana/fisiología , Proteínas de la Membrana Bacteriana Externa , Proteínas Portadoras , Streptococcus pyogenes/fisiología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , ADN Bacteriano/genética , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Infecciones Estreptocócicas/etiología , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidadRESUMEN
Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of alpha 5 beta 1:Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within alpha 5 beta 1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn(2+)-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg(2+)-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca(2+)-binding site. Taken together, our results imply that the ligand-binding capacity of alpha 5 beta 1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.
Asunto(s)
Calcio/farmacología , Adhesión Celular , Fibronectinas/metabolismo , Magnesio/farmacología , Manganeso/farmacología , Receptores de Fibronectina/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Línea Celular , Cromatografía de Afinidad , Femenino , Fibronectinas/efectos de los fármacos , Humanos , Cinética , Leucemia Eritroblástica Aguda , Leucocitos Mononucleares/fisiología , Ligandos , Magnesio/metabolismo , Manganeso/metabolismo , Placenta/fisiología , Embarazo , Ratas/inmunología , Receptores de Fibronectina/efectos de los fármacos , Receptores de Fibronectina/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.