Plasminogen Activation–Induced Pericellular Fibronectin Proteolysis Promotes Fibroblast Apoptosis

Abstract

Apoptosis of fibroblasts/myofibroblasts is a critical event in the resolution of tissue repair responses; however, mechanisms for the regulation of (myo)fibroblast apoptosis/survival remain unclear. In this study, we demonstrate counter-regulatory interactions between the plasminogen activation system and transforming growth factor-b1 (TGF-b1) in the control of fibroblast apoptosis. Plasmin treatment induced fibroblast apoptosis in a time-and dosedependent manner in association with proteolytic degradation of extracellular matrix proteins, as detected by the release of soluble fibronectin peptides. Plasminogen, which was activated to plasmin by fibroblasts, also induced fibronectin proteolysis and fibroblast apoptosis, both of which were blocked by a2-antiplasmin but not by inhibition of matrix metalloproteinase activity. TGF-b1 protected fibroblasts from apoptosis induced by plasminogen but not from apoptosis induced by exogenous plasmin. The protection from plasminogen-induced apoptosis conferred by TGF-b1 is associated with the up-regulation of plasminogen activator-1 (PAI-1) expression and inhibition of plasminogen activation. Moreover, lung fibroblasts from mice genetically deficient in PAI-1 lose the protective effect of TGF-b1 against plasminogen-induced apoptosis. These findings support a novel role for the plasminogen activation system in the regulation of fibroblast apoptosis and a potential role of TGF-b1/PAI-1 in promoting (myo)fibroblast survival in chronic fibrotic disorders.Keywords: myofibroblast; fibrosis; transforming growth factor-b; anoikis; plasminogen activator inhibitor 1Fibroblasts, versatile connective tissue cells and key effectors in wound-repair responses, are recruited to sites of tissue injury where extracellular matrix (ECM) components and soluble factors, such as transforming growth factor-b1 (TGF-b1), promote myofibroblast differentiation. Myofibroblasts secrete, organize, remodel, and contract the ECM, facilitating wound closure and reepithelialization (1). With the resolution of normal wound repair, fibroblast/myofibroblast numbers decrease substantially due to apoptosis (2). The physiologic stimuli that trigger fibroblast/ myofibroblast apoptosis, however, have not been determined (3). Failure of myofibroblast apoptosis is associated with pathologic wound repair characterized by tissue fibrosis (2, 4-6).TGF-b1 is a potent pro-fibrotic cytokine that promotes myofibroblast differentiation, migration, ECM synthesis, and resistance to apoptosis (1, 7-11). Studies from our laboratory have shown that TGF-b1-induced myofibroblast differentiation requires the integration of cell-matrix adhesion signals through activation of focal adhesion kinase (FAK) (8). Moreover, the coordinate activation of FAK and phosphatidylinositol-39-OH kinase/AKT (PI3K/AKT) through SMAD3 and p38 MAPKdependent pathways respectively confers fibroblast resistance to anoikis, a type of apoptosis induced by the loss of adhesion or adhesion-dependent signaling (9, 10, 12, 13). These findings suggest that TGF-b1 re...

SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of β-Catenin

“…SPARC has been shown to induce PAI-1 expression in aortic endothelial cells, but the mechanism was not elucidated (30). Our findings of elevated basal PAI-1 in IPF fibroblasts complement those of Horowitz et al (15), who found that TGF-␤ induces PAI-1 in lung fibroblasts, which confers resistance to anoikis and plasminogen-induced apoptosis. Our findings suggest that SPARC, but not TGF-␤, regulates basal expression of PAI-1; we did find, however, that TGF-␤-dependent induction of PAI-1 in control fibroblasts requires the TGF-␤1 receptor/ALK5 (Fig.…”

“…A recent study showed, for example, that TGF-␤ induction of PAI-1 in lung fibroblasts inhibits plasminogen-induced apoptosis of normal lung fibroblasts (15). Following our observation that IPF fibroblasts express significantly higher basal PAI-1 than control fibroblasts, we speculated that IPF fibroblasts would be more resistant than control fibroblasts to plasminogen-induced apoptosis.…”

“…Also, endothelin-1 and TGF-␤ appear to independently protect lung fibroblasts from apoptosis via these pathways (13,14). The same laboratory also showed that TGF-␤-mediated induction of plasminogen activator inhibitor-1 (PAI-1) protects lung fibroblasts from plasminogen-induced apoptosis (15). Several previous studies show that PAI-1 promotes fibrosis in lung and other tissues and that plasminogen activation is anti-fibrogenic (16,17).…”

“…We identified PAI-1 as a potential target because the balance of expression of plasminogen versus PAI-1 regulates matrix production and turnover during wound healing, PAI-1 expression is increased in IPF lung, deficiency in PAI-1 protects mice from bleomycin-induced fibrosis, and it has been shown to mediate resistance of lung fibroblasts to plasminogen-induced apoptosis in response to TGF-␤ and endothelin-1 (Ref. 15; reviewed in Ref. 17).…”

siRNA against plasminogen activator inhibitor-1 ameliorates bleomycin-induced lung fibrosis in rats

“…It is generally accepted that PAI-1 binding to uPA and the uPA receptor induces internalisation of the complex, thereby facilitating extracellular proteolysis as well as activation of growth factors and anti-apoptosis proteins [28,29] . Furthermore, these intermediates activate several signalling pathways.…”