Role of the Urokinase-Fibrinolytic System in Epithelial–Mesenchymal Transition during Lung Injury
2014; Elsevier BV; Volume: 185; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2014.08.027
ISSN1525-2191
AutoresAmarnath S. Marudamuthu, Yashodhar P. Bhandary, Shwetha K. Shetty, Jian Fu, Venkatachalem Sathish, Y. S. Prakash, Sreerama Shetty,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoAlveolar 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. 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. Idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases are characterized by destruction of lung architecture due to excessive deposition of extracellular matrix proteins by activated fibroblasts or myofibroblasts, leading to progressive dyspnea and loss of lung function.1Kim K.K. Kugler M.C. Wolters P.J. Robillard L. Galvez M.G. Brumwell A.N. Sheppard D. Chapman H.A. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.Proc Natl Acad Sci U S A. 2006; 103: 13180-13185Crossref PubMed Scopus (999) Google Scholar, 2Chapman H.A. Disorders of lung matrix remodeling.J Clin Invest. 2004; 113: 148-157Crossref PubMed Scopus (168) Google Scholar, 3Kalluri R. Neilson E.G. Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2059) Google Scholar The origins of myofibroblasts participating in the pathological remodeling of IPF lungs are not clear. Histopathological evaluation demonstrates that myofibroblasts accumulate in fibroblastic foci. Emerging evidence suggests that polarized type II alveolar epithelial (ATII) cells undergo epithelial–mesenchymal transitions (EMT) after lung injury. The ATII cells assume phenotypic changes such as increased migration, invasion, resistance to apoptosis, and production of elevated levels of extracellular matrix proteins4Konigshoff M. Kramer M. Balsara N. Wilhelm J. Amarie O.V. Jahn A. Rose F. Fink L. Seeger W. Schaefer L. Günther A. Eickelberg O. WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis.J Clin Invest. 2009; 119: 772-787PubMed Google Scholar, 5Zhong Q. Zhou B. Ann D.K. Minoo P. Liu Y. Banfalvi A. Krishnaveni M.S. Dubourd M. Demaio L. Willis B.C. Kim K.J. duBois R.M. Crandall E.D. Beers M.F. Borok Z. Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.Am J Respir Cell Mol Biol. 2011; 45: 498-509Crossref PubMed Scopus (147) Google Scholar and therefore serve as a source of myofibroblasts. Understanding the possible mechanisms contributing to EMT in ATII cells may help identify new targets to treat or at least limit fibrogenesis after lung injury.A number of molecular processes are involved in the initiation of EMT in ATII cells.5Zhong Q. Zhou B. Ann D.K. Minoo P. Liu Y. Banfalvi A. Krishnaveni M.S. Dubourd M. Demaio L. Willis B.C. Kim K.J. duBois R.M. Crandall E.D. Beers M.F. Borok Z. Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.Am J Respir Cell Mol Biol. 2011; 45: 498-509Crossref PubMed Scopus (147) Google Scholar Components of the fibrinolytic system such as urokinase-type plasminogen activator (uPA), uPA plasma membrane receptor (uPAR), and its major inhibitor, plasminogen activator inhibitor (PAI-1) are all elaborated by ATII cells. These proteins independently influence a broad range of biological processes germane to lung injury and its repair.6Shetty S. Bdeir K. Cines D.B. Idell S. Induction of plasminogen activator inhibitor-1 by urokinase in lung epithelial cells.J Biol Chem. 2003; 278: 18124-18131Crossref PubMed Scopus (30) Google Scholar However, their role in fibrogenesis via EMT is unclear. Recent publications using bleomycin (BLM)7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google Scholar and a passive cigarette smoke (PCS)8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar or adenovirus expressing constitutively active transforming growth factor β (Ad-TGF-β)1Kim K.K. Kugler M.C. Wolters P.J. Robillard L. Galvez M.G. Brumwell A.N. Sheppard D. Chapman H.A. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.Proc Natl Acad Sci U S A. 2006; 103: 13180-13185Crossref PubMed Scopus (999) Google Scholar, 9Huang W.T. Vayalil P.K. Miyata T. Hagood J. Liu R.M. Therapeutic value of small molecule inhibitor to plasminogen activator inhibitor-1 for lung fibrosis.Am J Respir Cell Mol Biol. 2012; 46: 87-95Crossref PubMed Scopus (59) Google Scholar exposure model of lung injury indicate that a coordinate increase in PAI-1 and a decrement in uPA by ATII cells promote lung injury and subsequent pulmonary fibrosis (PF). We also found that caveolin-1 scaffolding domain peptide (CSP) acts as a competitor to caveolin-1, restores expression of uPA and uPAR, and inhibits PAI-1 in ATII cells after lung injury. These changes prevent development of PF after lung injury.7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google Scholar Recent literature suggests that up to 30% to 50% of myofibroblasts may be derived via EMT during fibrogenesis.10Degryse A.L. Tanjore H. Xu X.C. Polosukhin V.V. Jones B.R. McMahon F.B. Gleaves L.A. Blackwell T.S. Lawson W.E. Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L442-L452Crossref PubMed Scopus (183) Google Scholar, 11Tanjore H. Xu X.C. Polosukhin V.V. Degryse A.L. Li B. Han W. Sherrill T.P. Plieth D. Neilson E.G. Blackwell T.S. Lawson W.E. Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis.Am J Respir Crit Care Med. 2009; 180: 657-665Crossref PubMed Scopus (318) Google Scholar, 12Radisky D.C. Kenny P.A. Bissell M.J. Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT?.J Cell Biochem. 2007; 101: 830-839Crossref PubMed Scopus (276) Google Scholar However, an in vivo genetic lineage tracing study reported by Rock et al13Rock J.R. Barkauskas C.E. Cronce M.J. Xue Y. Harris J.R. Liang J. Noble P.W. Hogan B.L. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.Proc Natl Acad Sci U S A. 2011; 108: E1475-E1483Crossref PubMed Scopus (670) Google Scholar contradicts these findings. Our objective in the current study is to elucidate the role of altered expression of uPA, uPAR, and PAI-1 after lung injury in EMT, and further evaluate whether reinstatement of baseline expression of uPA, uPAR, and PAI-1 by CSP intervention after lung injury reduces EMT in ATII cells.Materials and MethodsIsolation and Analysis of ATII Cells from IPF and COPD Lung TissuesIPF or chronic obstructive pulmonary disease (COPD), and control (without overt IPF or COPD) donor lung samples were collected from patients undergoing thoracic surgery at St. Mary's Hospital (Rochester, MN). Protocols were approved by institutional review boards of Mayo Clinic (Rochester, MN) and The UT Health Science Center at Tyler (Tyler, TX). ATII cells were isolated from the IPF, COPD, and control lung tissues, and aliquots were stained for inclusion bodies to assess the purity of cell preparation as we described recently.8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar The lysates from these cells were immunoblotted for expression of EMT markers. Lung sections from patients with IPF and COPD, diffused alveolar damage, or healthy donors were provided by the Lung Tissue Research Consortium of the National Heart, Lung, and Blood Institute or by the Department of Pathology at the UT Health Science Center at Tyler. These sections were subjected to immunohistochemical (IHC) analysis to assess changes in antigen levels for uPA, PAI-1, and EMT markers.BLM- and PCS-Induced Lung InjuryWild-type (WT) or uPA-, uPAR-, and PAI-1–deficient mice of C57BL/6 background were bred in our facilities or were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed according to approved protocols under the guidelines of the Animal Care and Use Committee of The UT Health Science Center at Tyler. For in vitro experiments, ATII cells were isolated from the lungs of uninjured mice as we reported elsewhere.7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google Scholar, 8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar The purities of ATII cell preparations were approximately 90% to 95%, based on lithium carbonate staining for inclusion bodies. These cells were treated with 40 μg/mL BLM with or without 10 nmol/L CSP or control peptide (CP), or 2 ng/mL purified TGF-β1 alone in Matrigel-coated culture dishes containing epithelial cell growth–supplemented alveolar epithelial cell medium (AEpiCM) from ScienCell Research Laboratories (Carlsbad, CA) for 3 days.7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google Scholar, 8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar, 14Corti M.1 Brody A.R. Harrison J.H. Isolation and primary culture of murine alveolar type II cells.Am J Respir Cell Mol Biol. 1996; 14: 309-315Crossref PubMed Scopus (274) Google Scholar, 15Dobbs L.G. Isolation and culture of alveolar type II cells.Am J Physiol. 1990; 258: L134-L147PubMed Google Scholar, 16Rice W.R. Conkright J.J. Na C.L. Ikegami M. Shannon J.M. Weaver T.E. Maintenance of the mouse type II cell phenotype in vitro.Am J Physiol Lung Cell Mol Physiol. 2002; 283: L256-L264PubMed Google Scholar, 17Meng X. Ezzati P. Wilkins J.A. Requirement of podocalyxin in TGF-beta induced epithelial mesenchymal transition.PLoS One. 2011; 6: e18715Crossref PubMed Scopus (58) Google Scholar For in vivo studies, mice were exposed to 2 U BLM per kg body weight for 3 days or PCS for 20 weeks with or without 18.75 mg of CSP or CP per kg body weight as described previously.7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google Scholar, 8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar ATII cells were isolated and used for Western blot analysis or real-time PCR analysis to assess changes in the expression of E-cadherin, ZO-1, collagen-I, and α-smooth muscle actin (α-SMA) protein and mRNA, respectively. Real-time PCR using primers for mouse E-cadherin, forward: 5′-AATGGCGGCAATGCAATCCCAAGA-3′ and reverse: 5′-TGCCACAGACCGATTGTGGAGATA-3′; mouse collagen-I, forward: 5′-CCAAGGGTAACAGCGGTGAA-3′ and reverse: 5′-CCTCGTTTTCCTTCTTCTCCG-3′; mouse ZO-1, forward: 5′-ATTCTGAAGAAATGATGAGA-3′ and reverse: 5′-TCCTGATTGACCACTTTTAA-3′; α-SMA, forward: 5′-GGCTCTGGGCTCTGTAAGG-3′ and reverse: 5′-CTCTTGCTCTGGGCTTCATC-3′; and β-actin, forward: 5′-CACCGCAGCTCGTAGCTCTTCTCCAGGG-3′ and reverse: 5′-CCAGCCATGTACGTTGCTATCCAG-3′. Lung sections were separately assessed for collagen-I, E-cadherin, α-SMA, and ZO-1 antigen levels by IHC analysis. Lung homogenates and bronchoalveolar lavage fluids of mice exposed to BLM with or without CSP or CP for 3 days were analyzed for active TGF-β by Western blot analysis. The findings were independently confirmed by enzyme-linked immunosorbent assay (ELISA) for active TGF-β.Effect of TGF-β Overexpression on ATII Cell EMT and PFATII cells isolated from WT mice were exposed to Ad-TGF-β in the presence or absence of 10 nmol/L CSP or CP as described above. ATII cells untreated or treated with empty adenovirus (Ad-EV) for 3 days were used as controls. For in vivo studies, WT mice were exposed to saline or Ad-EV or Ad-TGF-β (109 plaque forming units) through intranasal instillation as described earlier.9Huang W.T. Vayalil P.K. Miyata T. Hagood J. Liu R.M. Therapeutic value of small molecule inhibitor to plasminogen activator inhibitor-1 for lung fibrosis.Am J Respir Cell Mol Biol. 2012; 46: 87-95Crossref PubMed Scopus (59) Google Scholar After 24 hours, mice exposed to Ad-TGF-β were treated with or without 18.75 mg CSP or CP per kg body weight. ATII cells were isolated 3 days after instillation of Ad-TGF-β and analyzed for changes in EMT. Lung homogenates of WT mice were analyzed for total hydroxyproline contents 21 days after transduction with Ad-TGF-β to assess changes in PF as we described earlier.7Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Gyetko M.R. Idell S. Gharaee-Kermani M. Shetty R.S. Starcher B.C. Shetty S. Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L463-L473Crossref PubMed Scopus (59) Google ScholarEffect of p53-Binding uPA, uPAR, and PAI-1 mRNA 3′-UTR Sequences on BLM-Induced ATII Cell EMT in MiceThe competitive inhibition of p53 from binding to endogenous uPA, uPAR, and PAI-1 mRNAs in ATII cells by overexpression of p53-binding chimeric uPA, uPAR, and PAI-1 3′-untranslated region (UTR) sequences, concurrently restores uPA and uPAR, and inhibits PAI-1 expression without affecting BLM- or PCS-induced p53 in mice.8Shetty S.K. Bhandary Y.P. Marudamuthu A.S. Abernathy D. Velusamy T. Starcher B. Shetty S. Regulation of airway and alveolar epithelial cell apoptosis by p53-induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.Am J Respir Cell Mol Biol. 2012; 47: 474-483Crossref PubMed Scopus (38) Google Scholar, 18Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Ji H.L. Neuenschwander P.F. Boggaram V. Morris G.F. Fu J. Idell S. Shetty S. Regulation of lung injury and fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1.Am J Pathol. 2013; 183: 131-143Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Therefore, we exposed mice to an i.v. (through orbital plexus) injection of lentivirus vector containing SP-B promoter expressing p53-binding or nonbinding control chimeric sequences of uPA, uPAR, and PAI-1 3′-UTR mRNA, and the transduction efficiency was confirmed by expression of luciferase in ATII cells as described previously.18Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Ji H.L. Neuenschwander P.F. Boggaram V. Morris G.F. Fu J. Idell S. Shetty S. Regulation of lung injury and fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1.Am J Pathol. 2013; 183: 131-143Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Twenty-four hours after lentiviral transduction, these mice were exposed to BLM. ATII cells were isolated 72 hours after initiation of BLM injury and analyzed for EMT markers by Western blot analysis.Effect of Inhibition of uPA or PAI-1 Expression in PAI-1– and uPA-Deficient ATII Cells, Respectively, on EMTATII cells isolated from uninjured mice lacking PAI-1 expression were treated with lentiviral vector expressing uPA shRNA to inhibit baseline uPA expression. PAI-1–deficient ATII cells exposed to control shRNA or naive ATII cells were used as controls. Similarly, ATII cells isolated from uPA-deficient mice were exposed to BLM in the presence or absence of PAI-1 shRNA or control shRNA. The lysates from these mice were analyzed for uPA, E-cadherin, ZO-1, and α-SMA expression.Generation of Retroviral PlasmidRetrovirus vector (pLNCX) containing Src mutant Y416F cDNA was cotransfected with packaging plasmid pUMVC and auxiliary plasmid pCMV-VSV-G (Addgene, Cambridge, MA) using Lipofectamine 2000 (Life Technologies, Grand Island, NY) in 293T cells to obtain phage particles. The viral titers were measured using 293 T cells and later transduced ATII cells isolated from mouse lungs.Localization of Biotin-Labeled CSP in Mouse LungsTo determine the distribution of CSP in the mouse lungs, mice were exposed to saline or BLM for 72 hours through intranasal instillation. These mice were i.p. injected with biotin-labeled CSP. After 24 hours, mice were euthanized, and the lung sections were analyzed for biotin-labeled CSP using anti-biotin antibody.Statistical AnalysisThe statistical differences between various experimental conditions were analyzed by t-test and one way analysis of variance. P < 0.05 was considered significant.ResultsATII Cell EMT Is Associated with Increased Expression of PAI-1 and Concurrent Inhibition of uPA in IPF and COPD LungsATII cell damage precedes development of PF, and restoration of uPA and inhibition of PAI-1 expression prevents development of PF after fibrosing lung injury in animal models. Multiple recent studies indicate that EMT in ATII cells promotes fibrogenesis after lung injury. Therefore, we isolated ATII cells from the human lung tissues and stained for inclusion bodies to assess the purity of ATII cell preparation using lithium carbonate, which indicated purity approximately 90% to 95% (Figure 1A). Next, we tested the ATII cell lysates for changes in uPA and PAI-1 expression. ATII cells isolated from human IPF lungs showed a marked increase in PAI-1 and reduction in uPA expression compared to those extracted from histologically normal lungs (Figure 1B). ATII cells from IPF lungs showed suppression of epithelial cell markers (E-cadherin and ZO-1) expression, whereas mesenchymal cell markers (collagen-I and α-SMA) were increased compared to their baseline expressions found in control lungs. We also found similar changes in the expression of uPA, PAI-1, and markers of EMT in ATII cells from the lungs of patients with COPD. Consistent with the results of Western blot analysis of isolated ATII cells, IHC analyses of IPF and COPD lung sections showed increased collagen-I and inhibition of ZO-1 staining compared to their expression in control subjects (Figure 1C). Similar changes were also observed in lung sections of patients with diffused alveolar damage. This indicates a causal link between altered ATII cell collagen-I and ZO-1 expression, and EMT as a result of chronic lung injury.Effect of CSP on BLM-Induced EMT in ATII CellsBecause ATII cells from human IPF lungs showed increased EMT and PAI-1 with reduction in uPA expression, we isolated ATII cells from mouse lungs and exposed them to BLM for 72 hours and analyzed changes in the expression of E-cadherin, ZO-1, collagen-I, and α-SMA to assess EMT in vitro. Exposure of ATII cells to BLM suppressed expression of E-cadherin and ZO-1, while increasing collagen-I and α-SMA (Figure 2A). ATII cells treated with the profibrogenic cytokine, TGF-β, also revealed a significant increase in EMT compared to phosphate-buffered saline (PBS)-treated control ATII cells. Exposure of ATII cells to BLM with CSP restored E-cadherin and ZO-1 expression while significantly reducing the expression of collagen-I and α-SMA. However, the scrambled CP failed to affect BLM-induced EMT in murine ATII cells. Because treatment of mouse ATII cells with purified TGF-β protein increased EMT, we next transduced ATII cells with Ad-TGF-β in the presence or absence of CSP or CP and tested for changes in EMT markers. Ad-TGF-β increased EMT in ATII cells in comparison with those transduced with Ad-EV (Figure 2B). Further, treatment of ATII cells with CSP, but not CP, suppressed TGF-β–induced EMT. To determine whether in vitro culture condition alters phenotypes of ATII cells during the course of our experiments, we maintained control ATII cells isolated from uninjured mice in growth factor–supplemented AEpiCM medium for 0 to 3 days and analyzed for changes in surfactant proteins by Western blot analysis. ATII cells cultured in AEpiCM retained baseline SP-A, SP-B, and SP-C expression at least for 3 days, indicating preservation of cellular phenotypes (Figure 2C).Figure 2Inhibition of bleomycin (BLM)-induced epithelial–mesenchymal transitions (EMT) in ATII cells by caveolin-1 scaffolding domain peptide (CSP). A: ATII cells isolated from WT mice were treated with PBS or 40 μg/mL BLM alone or BLM with 10 nmol/L CSP or control peptide (CP) for 72 hours in culture dishes at 37°C. The lysates were immunoblotted for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin antigens. The lysates from ATII cells treated with 2 ng/mL transforming growth factor β (TGF-β) were used as a positive control for comparison. The densities of individual bands were normalized against the corresponding densities of β-actin. The fold changes in proteins are presented as a bar graph. Differences between PBS control and BLM or BLM and BLM+CSP groups are statistically significant. B: ATII cells isolated from WT mice were exposed to PBS, Ad-EV, or Ad-TGF-β with or without CSP or CP in culture dishes. After 72 hours, the lysates were analyzed for changes in EMT markers by Western blot analysis. C: ATII cells isolated from WT mice were cultured in Matrigel-coated plastic dishes for 0 to 3 days at 37°C. The lysates were later analyzed for changes in SP-A, SP-B, and SP-C expression by Western blot analysis. D: WT mice were exposed to saline or 2 U/kg body weight BLM through intranasal instillation. After 24 hours, mice exposed to BLM were i.p. injected with or without 18.75 mg/kg body weight of CSP or CP. Three days after BLM injury, ATII cells were isolated and the lysates were immunoblotted for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin. The densities of individual bands were measured and normalized against the corresponding densities of β-actin. The fold changes in proteins are presented as a bar graph. E: WT mice were exposed to ambient air or PCS from 40 cigarettes (approximately 90 mg/m3 total solid particulates) using a mechanical smoking chamber over a 2-hour period for 5 days per week. After 4 weeks of PCS exposure, mice exposed to PCS were i.p. injected with or without 18.75 mg/kg body weight of CSP or CP once every week for 4 more weeks. After 20 weeks of PCS exposure, ATII cells were isolated, and the lysates were tested for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1 and β-actin by Western blot analysis. F: Total RNA isolated from ATII cells of mice treated with saline, BLM, BLM+CSP, or CP were analyzed for changes in the expression of E-cadherin, collagen-I, ZO-1, and α-SMA mRNAs by quantitative real-time PCR. Changes in their expression levels were normalized to the corresponding levels of β-actin transcripts. The data were presented relative to that of saline-treated control groups. G: WT mice were exposed to saline, Ad-EV, or Ad-TGF-β by intranasal instillation. Twenty-four hours later, mice transduced with Ad-TGF-β were treated with or without CSP or CP. ATII cells were isolated from these mice 72 hours after exposure to TGF-β and analyzed for changes in EMT markers by Western blot analysis. H: WT mice treated with Ad-EV or Ad-TGF-β alone or Ad-TGF-β with CSP or CP as described in G were euthanized 21 days after initial transduction with Ad-TGF-β. The lung homogenates were analyzed for total hydroxylproline contents to assess changes in lung fibrosis. I: Mice were i.v. (via orbital plexus) injected with or without lentivirus expressing p53-binding or non–p53-binding control chimeric 3′-UTR sequences of uPA/uPAR/PAI-1 mRNA 3′-UTR as described elsewhere.14Corti M.1 Brody A.R. Harrison J.H. Isolation and primary culture of murine alveolar type II cells.Am J Respir Cell Mol Biol. 1996; 14: 309-315Crossref PubMed Scopus (274) Google Scholar Twenty-four hours later, the mice were exposed to BLM, and ATII cells were isolated 72 hours after inception of BLM injury. ATII cells isolated from mice exposed to saline were used as control for comparison. The lysates were tested for changes in the expression of collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin. ∗∗∗∗P < 0.0001 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we exposed the mice to BLM through intranasal instillation, and some mice were exposed to CSP or CP 24 hours after initiation of BLM injury. Saline-treated mice were used as controls. The lung sections from these mice were subjected to IHC analysis for changes in EMT markers in situ. Following BLM-induced injury, lung sections of mice showed a marked inhibition of ZO-1 antigen expression, whereas collagen-I antigen levels were markedly increased in the lung parenchyma (Supplemental Figure S1). Further, treatment of mice with CSP markedly reduced BLM-induced EMT, which was confirmed by the changes in ZO-1 or collagen-I expression, whereas the response of CP to BLM-induced
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