p53 Expression in Lung Fibroblasts Is Linked to Mitigation of Fibrotic Lung Remodeling.

Idiopathic pulmonary fibrosis (IPF) is a debilitating, incurable, and life-threatening disease. A cardinal feature of the pathogenesis of IPF is excessive extracellular matrix deposition attributable to proliferation of activated fibrotic lung fibroblasts (fLfs). To assess the underlying mechanism, we analyzed the status of the tumor suppressor protein p53 in fLfs from the lungs of IPF patients or mice with bleomycin-induced established PF. We report that basal expression of p53 is markedly reduced in fLfs. Forced expression of caveolin-1 in fLfs increased basal p53 and reduced profibrogenic proteins, including collagen-1. Transduction of fLfs with adenovirus expressing p53 reduced expression of these proteins. Conversely, inhibition of baseline p53 in control lung fibroblasts from lung tissues increased profibrogenic protein expression. Lung transduction of adenovirus expressing p53 reduced bleomycin-induced PF in wild-type or caveolin-1-deficient mice. Furthermore, treatment of fLfs or fibrotic lung tissues with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored p53 and reduced profibrogenic proteins. Treatment of wild-type mice with i.p. CSP or CSP7 resolved bleomycin-induced PF. These peptides failed to resolve PF in inducible conditional knockout mice lacking p53 in fLfs, indicating the induction of baseline fLf p53 as the basis of the antifibrotic effects.

p53 and miR-34a Feedback Promotes Lung Epithelial Injury and Pulmonary Fibrosis.

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.

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.

p53- and PAI-1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure.

We previously demonstrated that tumor suppressor protein p53 augments plasminogen activator inhibitor-1 (PAI-1) expression in alveolar epithelial cells (AECs) during chronic cigarette smoke (CS) exposure-induced lung injury. Chronic lung inflammation with elevated p53 and PAI-1 expression in AECs and increased susceptibility to and exacerbation of respiratory infections are all associated with chronic obstructive pulmonary disease (COPD). We recently demonstrated that preventing p53 from binding to the endogenous PAI-1 mRNA in AECs by either suppressing p53 expression or blockading p53 interactions with the PAI-1 mRNA mitigates apoptosis and lung injury. Within this context, we now show increased expression of the C-X-C chemokines (CXCL1 and CXCL2) and their receptor CXCR2, and the intercellular cellular adhesion molecule-1 (ICAM-1), in the lung tissues of patients with COPD. We also found a similar increase in lung tissues and AECs from wild-type (WT) mice exposed to passive CS for 20 wk and in primary AECs treated with CS extract in vitro. Interestingly, passive CS exposure of mice lacking either p53 or PAI-1 expression resisted an increase in CXCL1, CXCL2, CXCR2, and ICAM-1. Furthermore, inhibition of p53-mediated induction of PAI-1 expression by treatment of WT mice exposed to passive CS with caveolin-1 scaffolding domain peptide reduced CXCL1, CXCL2, and CXCR2 levels and lung inflammation. Our study reveals that p53-mediated induction of PAI-1 expression due to chronic CS exposure exacerbates lung inflammation through elaboration of CXCL1, CXCL2, and CXCR2. We further provide evidence that targeting this pathway mitigates lung injury associated with chronic CS exposure.

Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.

Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acute and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53-uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway.

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.

Development of a Triple-Negative Breast Cancer Leptomeningeal Disease Model in Zebrafish.

Leptomeningeal disease occurs when cancer cells migrate into the ventricles of the brain and spinal cord and then colonize the meninges of the central nervous system. The triple-negative subtype of breast cancer often progresses toward leptomeningeal disease and has a poor prognosis because of limited treatment options. This is due, in part, to a lack of animal models with which to study leptomeningeal disease. Here, we developed a translucent zebrafish casper (roy-/-; nacre-/-) xenograft model of leptomeningeal disease in which fluorescent labeled MDA-MB-231 human triple-negative breast cancer cells are microinjected into the ventricles of zebrafish embryos and then tracked and measured using fluorescent microscopy and multimodal plate reader technology. We then used these techniques to measure tumor area, cell proliferation, and cell death in samples treated with the breast cancer drug doxorubicin and a vehicle control. We monitored MDA-MB-231 cell localization and tumor area, and showed that samples treated with doxorubicin exhibited decreased tumor area and proliferation and increased apoptosis compared to control samples.

Regulation of airway and alveolar epithelial cell apoptosis by p53-Induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.

Regulation of urokinase expression at the posttranscription level by lung epithelial cells.

Urokinase-type plasminogen activator (uPA) is expressed by lung epithelial cells and regulates fibrin turnover and epithelial cell viability. PMA, LPS, and TNF-alpha, as well as uPA itself, induce uPA expression in lung epithelial cells. PMA, LPS, and TNF-alpha induce uPA expression through increased synthesis as well as stabilization of uPA mRNA, while uPA increases its own expression solely through uPA mRNA stabilization. The mechanism by which lung epithelial cells regulate uPA expression at the level of mRNA stability is unclear. To elucidate this process, we sought to characterize protein-uPA mRNA interactions that regulate uPA expression. Regulation of uPA at the level of mRNA stability involves the interaction of a ~40 kDa cytoplasmic-nuclear shuttling protein with a 66 nt uPA mRNA 3'UTR sequence. We purified the uPA mRNA 3'UTR binding protein and identified it as ribonucleotide reductase M2 (RRM2). We expressed recombinant RRM2 and confirmed its interaction with a specific 66 nt uPA 3'UTR sequence. Immunoprecipitation of cell lysates with anti-RRM2 antibody and RT-PCR for uPA mRNA confirmed that RRM2 binds to uPA mRNA. Treatment of Beas2B cells with uPA or LPS attenuated RRM2-endogenous uPA mRNA interactions, while overexpression of RRM2 inhibited uPA protein and mRNA expression through destabilization of uPA mRNA. LPS exposure of lung epithelial cells translocates RRM2 from the cytoplasm to the nucleus in a time-dependent manner, leading to stabilization of uPA mRNA. This newly recognized pathway could influence uPA expression and a broad range of uPA-dependent functions in lung epithelial cells in the context of lung inflammation and repair.

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.

Post-transcriptional regulation of plasminogen activator inhibitor type-1 expression in human pleural mesothelial cells.

The plasminogen activator inhibitor type-1 (PAI-1) effectively blocks the activities of free and receptor-bound urokinase-type plasminogen activator. Incubation of cultured human pleural mesothelial (Met5A) cells with TGF-beta increased PAI-1 protein. TGF-beta, phorbol myristate acetate, and the translation inhibitor cycloheximide induced PAI-1 mRNA and slowed its degradation, suggesting that PAI-1 mRNA could be regulated by interaction of a PAI-1 binding protein (PAI-1 mRNABp) with PAI-1 mRNA. We found that an approximately 60 kD cytoplasmic PAI-1 mRNABp is detectable in cytoplasmic extracts of MeT5A human pleural mesothelial and malignant mesothelioma cells. The PAI-1 mRNABp specifically binds to a 33-nt sequence in the 3' untranslated region of PAI-1 mRNA. Insertion of this 33-nt sequence destabilizes otherwise stable beta-globin mRNA, indicating that the binding sequence accelerates decay of endogenous PAI-1 mRNA. Competitive inhibition by overexpression of the 33-nt binding sequence in MeT5A cells reduced PAI-1 mRNA decay and increased PAI-1 protein and mRNA expression, indicating that the PAI-1 mRNABp destabilizes PAI-1 mRNA by its interaction with the endogenous 33-nt binding sequence. Incubation of Met5A cells with TGF-beta attenuated the interaction of the PAI-1 mRNABp with the 33-nt sequence. By conventional and affinity purification, we isolated the PAI-1 mRNABp and confirmed its identity as 6-phospho-d-gluconate-NADP oxidoreductase, which specifically interacts with the full-length and the 33-nt sequence of the PAI-1 mRNA 3' untranslated region. This newly recognized pathway could influence expression of PAI-1 by mesothelial or mesothelioma cells at the level of mRNA stability in the context of pleural inflammation or malignancy.

Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.

Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.