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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 α5β1 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 β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 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.

Transforming growth factor (TGF)-β1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-β1-induced myofibroblast differentiation in vitro and in vivo. TGF-β1 can induce α-smooth muscle actin (α-SMA) expression in the presence or absence of FAK; however, TGF-β1-induced α-SMA expression is reduced (~73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-β1-induced α-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38MAPKis the dominant mediator of TGF-β1-induced α-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-β1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in α-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis. [ABSTRACT FROM AUTHOR]

We have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated in either soft agar or in the C.B.17 severe combined immunodeficiency (scid) mouse brain. We therefore determined the effect of FAK on cell cycle progression in these cells. We found that overexpression of wild-type FAX promoted exit from G1 in monolayer cultures of glioblastoma cells, enhanced the expression of cyclins D1 and E while reducing the expression of p27Kip1 and p21Waf1 and enhanced the kinase activity of the cyclin D1-cyclin-dependent kinase-4 (cdk4) complex. Transfection of the monolayers with a FAX molecule in which the autophosphorylation site is mutated (FAK397F) inhibited exit from G1 and reduced the expression of cyclins D1 and E while enhancing the expression of p27Kip1 and p21Waf1. Small interfering RNA (siRNA)-mediated down-regulation of cyclin D1 inhibited the enhancement of cell cycle progression observed on expression of wild-type FAX, whereas siRNA-mediated down-regulation of cyclin E had no effect. siRNA-mediated down-regulation of p27Kip1 overcame the inhibition of cell cycle progression observed on expression of FAK397F, whereas down-regulation of p21Waf1 had no effect. These results were confirmed in vivo in the scid mouse brain xenograft model in which propagation of glioblastoma cells expressing FAK397F resulted in a 50% inhibition of tumor growth and inhibited exit from G1. Taken together, our results indicate that FAX promotes proliferation of glioblastoma cells by enhancing exit from G1 through a mechanism that involves cyclin D1 and p27Kip1. [ABSTRACT FROM AUTHOR]

Enhanced expression of both integrin α[sub v]β[sub 3] and platelet-derived growth factor receptor (PDGFr) has been described in glioblastoma tumors. We therefore explored the possibility that integrin α[sub v]β[sub 3] cooperates with PDGFr to promote cell migration in glioblastoma cells, and extended the study to identify the Src family members that are activated on PDGF stimulation. Glioblastoma cells utilize integrins α[sub v]β[sub 3] and α[sub v]β[sub 5] to mediate vitronectin attachment. We found that physiologic PDGF stimulation (83 pM, 10 min) of vitronectin-adherent cells promoted the specific recruitment of integrin α[sub v]B[sub 3]-containing focal adhesions to the cell cortex and α[sub v]β[sub 3]-mediated cell motility. Analysis of PDGFr immunoprecipitates indicated an association of the PDGFr β with integrin α[sub v]β[sub 3], but not integrin α[sub v]β[sub 5]. Cells plated onto collagen or laminin, which engage different integrins, exhibited significantly less migration on PDGF stimulation, indicating a cooperation of α[sub v]β[sub 3] and the PDGFr β in glioblastoma cells that promotes migration. Further analysis of the cells plated onto vitronectin indicated that PDGF stimulation caused an increase in Src kinase activity, which was associated with integrin α[sub v]β[sub 3]. In the vitronectin-adherent cells, Lyn was associated preferentially with α[sub v]β[sub 3] both in the presence and absence of PDGF stimulation. In contrast, Fyn was associated with both α[sub v]β[sub 3] and α[sub v]β[sub 5]. Moreover, PDGF stimulation increased the activity of Lyn, but not Fyn, in vitronectin-adherent cells, and the activity of Fyn, but not Lyn, in laminin-adherent cells. Using cells attached to mAb anti-α[sub v]β[sub 3] or mAb anti-integrin α[sub 6], we confirmed the activation of specific members of the Src kinase family with PDGF stimulation. Down-regulation of Lyn expression by siRNA significantly inhibited the cell migration mediated by integrin α[sub v]β[sub 3] in PDGF-stimulated cells, demonstrating the PDGFr β cooperates with integrin α[sub v]β[sub 3] in promoting the motility of vitronectin-adherent glioblastoma cells through a Lyn kinase-mediated pathway. Notably, the data indicate that engagement of different integrins alters the identity of the Src family members that are activated on stimulation with PDGF. [ABSTRACT FROM AUTHOR]