Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury.

Alveolar type II epithelial (ATII) cell injury precedes development of pulmonary fibrosis. Mice lacking urokinase-type plasminogen activator (uPA) are highly susceptible, whereas those deficient in plasminogen activator inhibitor (PAI-1) are resistant to lung injury and pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) has been considered, at least in part, as a source of myofibroblast formation during fibrogenesis. However, the contribution of altered expression of major components of the uPA system on ATII cell EMT during lung injury is not well understood. To investigate whether changes in uPA and PAI-1 by ATII cells contribute to EMT, ATII cells from patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, and mice with bleomycin-, transforming growth factor β-, or passive cigarette smoke-induced lung injury were analyzed for uPA, PAI-1, and EMT markers. We found reduced expression of E-cadherin and zona occludens-1, whereas collagen-I and α-smooth muscle actin were increased in ATII cells isolated from injured lungs. These changes were associated with a parallel increase in PAI-1 and reduced uPA expression. Further, inhibition of Src kinase activity using caveolin-1 scaffolding domain peptide suppressed bleomycin-, transforming growth factor β-, or passive cigarette smoke-induced EMT and restored uPA expression while suppressing PAI-1. These studies show that induction of PAI-1 and inhibition of uPA during fibrosing lung injury lead to EMT in ATII cells.

Marudamuthu AS ，Bhandary YP ，Shetty SK ，Fu J ，Sathish V ，Prakash Y ，Shetty S ... -  《-》

Regulation of lung injury and fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1.

Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.

Bhandary YP ，Shetty SK ，Marudamuthu AS ，Ji HL ，Neuenschwander PF ，Boggaram V ，Morris GF ，Fu J ，Idell S ，Shetty S ... -  《-》

Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.

Bhandary YP ，Shetty SK ，Marudamuthu AS ，Gyetko MR ，Idell S ，Gharaee-Kermani M ，Shetty RS ，Starcher BC ，Shetty S ... -  《-》

Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically "normal" lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF.

Marudamuthu AS ，Shetty SK ，Bhandary YP ，Karandashova S ，Thompson M ，Sathish V ，Florova G ，Hogan TB ，Pabelick CM ，Prakash YS ，Tsukasaki Y ，Fu J ，Ikebe M ，Idell S ，Shetty S ... -  《-》

Regulation of p53-mediated changes in the uPA-fibrinolytic system and in lung injury by loss of surfactant protein C expression in alveolar epithelial cells.

Pulmonary surfactant protein C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross-talk and apoptosis of AECs. Therefore, we inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in the uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, whereas it increased both AEC p53 and apoptosis in wild-type and uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2 and their receptor CXCR2 as well as ICAM-1 expression, which is indicative of a proinflammatory response. Overexpression of p53-binding 3'-UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Furthermore, caveolin-1 expression and phosphorylation were increased in AECs, indicating an intricate link between caveolin-1 and Src kinase-mediated cell signaling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1, and p53 in the regulation of AEC apoptosis after injury was also determined in knockout mice.

Puthusseri B ，Marudamuthu A ，Tiwari N ，Fu J ，Idell S ，Shetty S ... -  《-》