Plasminogen activator inhibitor-1 regulates integrin alphavbeta3 expression and autocrine transforming growth factor beta signaling.
Fibrosis is characterized by elevated transforming growth factor beta (TGFbeta) 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 alphavbeta3 from the cell surface. Since increased alphavbeta3 expression correlates with increased TGFbeta signaling, we hypothesized that aberrant PAI-1-mediated alphavbeta3 endocytosis could initiate an autocrine loop of TGFbeta activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), alphavbeta3 endocytosis was reduced by approximately 75%, leaving alphavbeta3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFbeta 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 TGFbeta activity that correlated with an increase in alphavbeta3 and TGFbeta receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFbeta activation, as evidenced by a mere 25% reduction in TGFbeta activity when plasmin was inhibited. Treatment of cells with an alphavbeta3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFbeta inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFbeta by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and beta3 overexpression led to an increase in TGFbeta 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 alphavbeta3 integrin is critical for the control of TGFbeta signaling and the prevention of fibrotic disease.
Pedroja
Benjamin S
BS
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Kang
Leah E
LE
Imas
Alex O
AO
Carmeliet
Peter
P
Bernstein
Audrey M
AM
D000818:Animals; D019898:Autocrine Communication; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D005341:Fibrinolysin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008907:Mink; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D018076:DNA, Complementary; D039302:Integrin alphaVbeta3; D017395:Plasminogen Activator Inhibitor 1; D018125:Receptors, Transforming Growth Factor beta; D055293:Receptors, Urokinase Plasminogen Activator; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D005341:Fibrinolysin