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1.
J Biol Chem ; 289(18): 12791-804, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24644284

RESUMO

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5ß1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with ß1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5ß1 integrin and uPAR drive the translocation of α5ß1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.


Assuntos
Movimento Celular , Fibroblastos/metabolismo , Integrina alfa5beta1/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Western Blotting , Caveolinas/genética , Caveolinas/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibronectinas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Integrina alfa5beta1/genética , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Interferência de RNA , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Índice de Gravidade de Doença , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
2.
Am J Physiol Lung Cell Mol Physiol ; 297(1): L97-108, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19411312

RESUMO

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.


Assuntos
Movimento Celular , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Integrinas/metabolismo , Pulmão/citologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Animais , Bovinos , Adesão Celular , Extensões da Superfície Celular/metabolismo , Colágeno/metabolismo , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Fibrose/patologia , Humanos , Camundongos , Ligação Proteica , Subunidades Proteicas/metabolismo , Transporte Proteico , Vitronectina/metabolismo , Cicatrização
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