The Receptor for Urokinase-type Plasminogen Activator Regulates Fibronectin Matrix Assembly in Human Skin Fibroblasts

Abstract

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of ␣ 5 ␤ 1 -dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the ␤ 1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in ␣ 5 ␤ 1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6 -10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the ␤ 1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the ␣ 5 ␤ 1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.

PAI1 stimulates assembly of the fibronectin matrix in osteosarcoma cells through crosstalk between the αvβ5 and α5β1 integrins

“…PAI1 and uPAR have been shown to synergistically stimulate fibronectin polymerization (Monaghan et al, 2004;Monaghan-Benson and McKeown-Longo, 2006;Vial et al, 2006). In the current study, we show that PAI1 stimulates matrix assembly by activating a previously undescribed crosstalk between the αvβ5 and α5β1 integrins.…”

“…Activation of β1 integrin was assessed by ELISA using either the HUTS-4 or the 9EG7 antibody as described previously (Monaghan et al, 2004). These antibodies recognize the activated conformation of the β1 integrin.…”

Extracellular matrix dynamics in development and regenerative medicine

“…1). The uPAR ligand P25 peptide, through its association with uPAR, stimulates ␣5␤1 activity and FN fibrillogenesis in human skin fibroblasts in a Src-kinase-and epidermal growth factor receptor (EGFR)-dependent fashion (Monaghan-Benson and McKeown-Longo, 2006;Monaghan et al, 2004). In a separate study, Src, together with its substrate paxillin, also promoted prolonged integrin activation and thereby enhanced FN-matrix assembly and maintenance (Wierzbicka-Patynowski et al, 2007).…”

Regulation of urokinase receptor function and pericellular proteolysis by the integrin α5β1

“…uPAR and α 5 β 1 have been shown to co-imunoprecipitate in certain tumour cells (16), and we have previously observed this in the presence of CD82 (12,20); although not here in α5CHO cells. Indirect evidence for uPAR-α 5 β 1 interactions comes from observations on the effect of uPAR on various aspects of α 5 β 1 function, including signalling, migration and fibronectin matrix assembly (11,12,(16)(17)(18)(19)35). In many of these examples, uPA binding to uPAR is thought to promote the interaction with α 5 β 1 .…”

“…Degradation of ECM components by plasmin impacts on the highly regulated interactions between the ECM and the cytoskeleton, which are mediated through cell surface receptors of the integrin family. In addition to regulating plasmin generation, uPAR is also able to alter how cells interact with the ECM by affecting integrin function (8)(9)(10)(11)(12) and by direct interactions with vitronectin (13)(14)(15). The integrin α 5 β 1 is of particular interest as putative interactions between this integrin and uPAR have also been linked to uPAR-mediated signalling events (16)(17)(18)(19).…”