Plasminogen activator inhibitor-1 regulates integrin αvβ3 expression and autocrine transforming growth factor β signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (Gpen-GRGD) led to a more profound (59%) TGFβ inhibition; a non-specific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ 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 αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.