Plasminogen Activator Inhibitor-1 Regulates Integrin v 3 Expression and Autocrine Transforming Growth Factor Signaling*

Fibrosis is characterized by elevated transforming growth factor (TGF ) signaling, resulting in extracellular matrix accumulation and increasedPAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin v 3 from the cell surface. Since increased v 3 expression correlates with increased TGF signaling,wehypothesized that aberrant PAI-1-mediated v 3 endocytosis could initiate an autocrine loop of TGF activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), v 3 endocytosis was reduced by 75%, leaving v 3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGF signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9fold increase inTGF activity that correlatedwith an increase in v 3 and TGF receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGF activation, as evidenced by a mere 25% reduction in TGF activity when plasmin was inhibited. Treatment of cells with an v 3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGF inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGF by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and 3 overexpression led to an increase in TGF activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of v 3 integrin is critical for the control of TGF signaling and the prevention of fibrotic disease.