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.

Treatment of human pleural mesothelioma (MS-1) cells with phorbol myristate acetate (PMA) and cycloheximide results in 17-and 10-fold, respectively, increases in steady-state expression of urokinase-type plasminogen activator receptor (uPAR) mRNA. Studies of transcriptional inhibition by actinomycin D showed four-and sixfold extensions of uPAR mRNA half-life in MS-1 cells treated with PMA and cycloheximide, respectively, suggesting that uPAR gene expression involves a posttranscriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 50-kDa uPAR mRNA binding protein (uPAR mRNABp) that selectively bound to a 51-nucleotide (nt) fragment of mRNA corresponding to the uPAR coding region. We investigated the possibility that this 51-nt protein binding fragment of uPAR mRNA contains regulatory information for message stability. Chimeric ␤-globin/uPAR/␤-globin mRNA containing the 51-nt protein binding fragment was able to destabilize otherwise stable ␤-globin mRNA. Conversely, a control chimeric ␤-globin/uPAR/␤-globin mRNA containing a 51-nt fragment of the uPAR coding region that does not bind uPAR mRNABp was stable under identical conditions. Binding of uPAR mRNABp to uPAR mRNA was abolished after treatment with cycloheximide and rapidly down-regulated by PMA. These data suggest that the 51-nt protein binding fragment of uPAR mRNA may be involved in mRNA turnover as well as in cycloheximideinduced uPAR message stabilization. Our results indicate a novel mechanism of uPAR gene regulation in which cis elements within a 51-nt coding region interact with a uPAR mRNABp to regulate uPAR message stability.

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.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. The pathogenesis of interstitial lung diseases, including its most common form, IPF, remains poorly understood. Alveolar epithelial cell (AEC) apoptosis, proliferation, and accumulation of myofibroblasts and extracellular matrix deposition results in progressive loss of lung function in IPF. We found induction of tumor suppressor protein, p53, and apoptosis with suppression of urokinase-type plasminogen activator (uPA) and the uPA receptor in AECs from the lungs of IPF patients, and in mice with bleomycin, cigarette smoke, silica, or sepsis-induced lung injury. Treatment with the caveolin-1 scaffolding domain peptide (CSP) reversed these effects. Consistent with induction of p53, AECs from IPF lungs or mice with diverse types of lung injuries showed increased p53 acetylation and miR-34a expression with reduction in Sirt1. This was significantly reduced after treatment of wild-type mice with CSP, and uPA-deficient mice were unresponsive. Bleomycin failed to induce miR-34a in p53- or plasminogen activator inhibitor-1 (PAI-1)-deficient mice. CSP-mediated inhibition of miR-34a restored Sirt1, suppressed p53 acetylation and apoptosis in injured AECs, and prevented pulmonary fibrosis (PF). AEC-specific suppression of miR-34a inhibited bleomycin-induced p53, PAI-1, and apoptosis and prevented PF, whereas overexpression of precursor-miR-34a increased p53, PAI-1, and apoptosis in AECs of mice unexposed to bleomycin. Our study validates p53-miR-34a feedback as a potential therapeutic target in PF.

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as 125 I-uPA binding in Beas2B lung epithelial cells in a time-and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.

Urokinase (uPA) interacts with its receptor (uPAR) to promote proteolysis and tumor migration, functions of potential importance in the pathogenesis of malignant mesothelioma. Immunohistochemistry of human malignant mesothelioma tissue and mesothelioma cells (MS-1) showed that mesothelioma cells express uPAR. We isolated uPAR from MS-1 cells by metabolic labeling and showed that it could be induced by phorbol myristate acetate (PMA), lipopolysaccharide (LPS), a transforming growth factor-beta (TGF-beta) or tumor necrosis factor-alpha (TNF-alpha). Experiments with MS-1 cells showed that uPA binding was saturable, specific, and reversible with a mean dissociation constant (Kd) of 5.4 +/- 1.1 nM. Binding was inhibited by a blocking antibody to uPAR and by the uPA amino-terminal fragment (ATF), but not by low molecular weight uPA. uPAR expression was regulated transcriptionally and translationally; antisense oligonucleotides blocked expression of uPAR protein. Plasminogen activator inhibitor-1 (PAI-1) inhibited PA activity of preformed uPA/uPAR complexes and increased cycling of the receptor from the cell surface. Stimulation of subconfluent MS-1 cells by high molecular weight or recombinant uPA, but not ATF or low molecular weight fragment, caused concentration-dependent incorporation of [3H]thymidine. These data indicate a novel mechanism by which malignant mesothelioma cells localize pericellular proteolysis and concurrently regulate tumor cell proliferation.

We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. Although increased circulating levels of uPA are present in endotoxemia and sepsis, conditions in which activated neutrophils contribute to the development of acute organ dysfunction, the ability of uPA to participate directly in LPS-induced neutrophil activation has not been examined. In the present experiments, we show that uPA can enhance activation of neutrophils exposed to submaximal stimulatory doses of LPS. In particular, uPA increased LPS-induced activation of intracellular signaling pathways, including Akt and c-Jun N-terminal kinase, nuclear translocation of the transcriptional regulatory factor NF-κB, and expression of proinflammatory cytokines, including IL-1β, macrophage-inflammatory protein-2, and TNF-α. There was no effect of uPA on LPS-induced activation of p38 mitogen-activated protein kinase in neutrophils. Transgenic mice unable to produce uPA (uPA−/−) were protected from endotoxemia-induced lung injury, as determined by development of lung edema, pulmonary neutrophil accumulation, lung IL-1β, macrophage-inflammatory protein-2, and TNF-α cytokine levels. These results demonstrate that uPA can potentiate LPS-induced neutrophil responses and also suggest that such effects are sufficiently important in vivo to play a major contributory role in neutrophil-mediated inflammatory responses, such as the development of acute lung injury.