Rashmi S. Shetty

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53-/- cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53-/- cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3′-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3′-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3′-UTR sequence, and insertion of the p53-binding sequence into β-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296–393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

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

Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.

Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA.

ABSTRACT We found that p53-deficient (p53−/−) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53−/− cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3′ untranslated region (3′UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3′UTR sequence, and insertion of the p53 binding sequence into β-globin mRNA destabilized β-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric β-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells.

Induction of Tissue Factor by Urokinase in Lung Epithelial Cells and in the Lungs

RATIONALE Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates. OBJECTIVES This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs). METHODS Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA(-/-)) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity. MEASUREMENTS AND MAIN RESULTS uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA(-/-) mice exposed to LPS failed to induce TF. CONCLUSIONS uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.