p53 and miR-34a Feedback Promotes Lung Epithelial Injury and Pulmonary Fibrosis
2017; Elsevier BV; Volume: 187; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2016.12.020
ISSN1525-2191
AutoresShwetha K. Shetty, Nivedita Tiwari, Amarnath S. Marudamuthu, Bijesh Puthusseri, Yashodhar P. Bhandary, Jian Fu, Jeffrey Levin, Steven Idell, Sreerama Shetty,
Tópico(s)Circular RNAs in diseases
ResumoIdiopathic 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. 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. The tumor suppressor protein, p53, induces cell cycle arrest, apoptosis, senescence, and innate immunity. Induction of p53 occurs in response to DNA-damaging agents to protect against carcinogenesis.1Lakin N.D. Jackson S.P. Regulation of p53 in response to DNA damage.Oncogene. 1999; 18: 7644-7655Crossref PubMed Scopus (789) Google Scholar, 2Sui X. Han W. Pan H. p53-Induced autophagy and senescence.Oncotarget. 2015; 6: 11723-11724Crossref PubMed Scopus (15) Google Scholar, 3Granja A.G. Nogal M.L. Hurtado C. Salas J. Salas M.L. Carrascosa A.L. Revilla Y. Modulation of p53 cellular function and cell death by African swine fever virus.J Virol. 2004; 78: 7165-7174Crossref PubMed Scopus (46) Google Scholar, 4Lowe S.W. Schmitt E.M. Smith S.W. Osborne B.A. Jacks T. p53 Is required for radiation-induced apoptosis in mouse thymocytes.Nature. 1993; 362: 847-849Crossref PubMed Scopus (2764) Google Scholar Distinct signaling mechanisms have evolved to induce p53 through posttranslational stabilization. Cell cycle arrest because of elaboration of p53 can be transient or permanent as in the case of senescence and promote alveolar epithelial cell (AEC) apoptosis, especially if the damage is severe and beyond repair. The outcomes are decided by the extent of p53 protein accumulation, with lower levels promoting cell cycle arrest and higher levels inducing apoptosis. The cellular responses of p53 are presumed to be mediated by its ability to activate or repress downstream target genes at the transcriptional or posttranscriptional levels. These include genes that express proteins or noncoding RNAs responsible for cell cycle arrest, proliferation, cell signaling, senescence, and apoptosis.5Felton-Edkins Z.A. Kenneth N.S. Brown T.R.P. Daly N.L. Gomez-Roman N. Grandori C. Eisenman R.N. White R.J. Direct regulation of RNA polymerase III transcription by RB, p53 and c-Myc.Cell Cycle. 2003; 2: 180-183Crossref Scopus (90) Google Scholar, 6Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. Menssen A. Meister G. Hermeking H. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest.Cell Cycle. 2007; 6: 1586-1593Crossref PubMed Scopus (808) Google Scholar, 7Xi Y. Shalgi R. Fodstad O. Pilpel Y. Ju J. Differentially regulated micro-RNAs and actively translated messenger RNA transcripts by tumor suppressor p53 in colon cancer.Clin Cancer Res. 2006; 12: 2014-2024Crossref PubMed Scopus (186) Google Scholar, 8Werner H. Karnieli E. Rauscher F.J. LeRoith D. Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.Proc Natl Acad Sci. 1996; 93: 8318-8323Crossref PubMed Scopus (337) Google Scholar, 9Huarte M. Guttman M. Feldser D. Garber M. Koziol M.J. Kenzelmann-Broz D. Khalil A.M. Zuk O. Amit I. Rabani M. Attardi L.D. Regev A. Lander E.S. Jacks T. Rinn J.L. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response.Cell. 2010; 142: 409-419Abstract Full Text Full Text PDF PubMed Scopus (1709) Google Scholar Lung injury due to exposure to DNA-damaging agents, such as bleomycin (BLM), cigarette smoke (CS), silica, or radiation, rapidly induces p53 expression in AECs.10Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Midde K.K. Ji H.-L. Shams H. Subramaniam R. Fu J. Idell S. Shetty S. Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.PLoS One. 2015; 10: e0123187Crossref Scopus (26) Google Scholar, 11Shetty 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 (41) Google Scholar, 12Bhandary 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 (62) Google Scholar, 13Bhandary 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 (66) Google Scholar, 14Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Fu J. Pinson B.M. Levin J. Shetty S. Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.Toxicol Appl Pharmacol. 2015; 283: 92-98Crossref PubMed Scopus (21) Google Scholar, 15Hickman A.W. Jaramillo R.J. Lechner J.F. Johnson N.F. α-Particle-induced p53 protein expression in a rat lung epithelial cell strain.Cancer Res. 1994; 54: 5797-5800PubMed Google Scholar Likewise, hypoxia and hyperoxia augment p53 expression in AECs and other lung resident cells.16O'Reilly M.A. Staversky R.J. Stripp B.R. Finkelstein J.N. Exposure to hyperoxia induces p53 expression in mouse lung epithelium.Am J Respir Cell Mol Biol. 1998; 18: 43-50Crossref PubMed Scopus (102) Google Scholar, 17Mantell L.L. Lee P.J. Signal transduction pathways in hyperoxia-induced lung cell death.Mol Genet Metab. 2000; 71: 359-370Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 18Sermeus A. Michiels C. Reciprocal influence of the p53 and the hypoxic pathways.Cell Death Dis. 2011; 2: e164Crossref PubMed Scopus (202) Google Scholar Marked induction of p53 leads to apoptosis in AECs and subsequent activation and overgrowth of activated fibroblasts, in turn promoting fibrogenesis and the development of pulmonary fibrosis (PF).13Bhandary 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 (66) Google Scholar, 19Sutherland L.M. Edwards Y.S. Murray A.W. Alveolar type II cell apoptosis.Comp Biochem Physiol A Mol Integr Physiol. 2001; 129: 267-285Crossref PubMed Scopus (37) Google Scholar Consistent with these observations, AECs surrounding the fibrotic foci in the lungs of patients with idiopathic pulmonary fibrosis (IPF) exhibit increased apoptosis as well as increased p53 and plasminogen activator inhibitor-1 (PAI-1) expression.20Marudamuthu A.S. Bhandary Y.P. Shetty S.K. Fu J. Sathish V. Prakash Y. Shetty S. Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury.Am J Pathol. 2015; 185: 55-68Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 21Nakashima N. Kuwano K. Maeyama T. Hagimoto N. Yoshimi M. Hamada N. Yamada M. Nakanishi Y. The p53-Mdm2 association in epithelial cells in idiopathic pulmonary fibrosis and non-specific interstitial pneumonia.J Clin Pathol. 2005; 58: 583-589Crossref PubMed Scopus (29) Google Scholar, 22Marudamuthu A.S. Shetty S.K. Bhandary Y.P. Karandashova S. Thompson M. Sathish V. Florova G. Hogan T.B. Pabelick C.M. Prakash Y.S. Tsukasaki Y. Fu J. Ikebe M. Idell S. Shetty S. Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.J Biol Chem. 2015; 290: 9428-9441Crossref PubMed Scopus (38) Google Scholar Similarly, large numbers of apoptotic AECs are detected in the lungs of mice with well-established PF long after (14 or 21 days) inception of BLM-induced fibrosing lung injury.12Bhandary 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 (62) Google Scholar These observations suggest that AECs continuously die in fibrotic lungs and are replaced by activated myofibroblasts or fibrotic lung fibroblasts, leading to destruction of lung architecture and progressive loss of lung function. AECs of patients with chronic obstructive pulmonary disease, emphysema, acute lung injury, acute respiratory distress syndrome, and diffuse alveolar damage characteristically exhibit increased AEC apoptosis and p53 expression,10Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Midde K.K. Ji H.-L. Shams H. Subramaniam R. Fu J. Idell S. Shetty S. Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.PLoS One. 2015; 10: e0123187Crossref Scopus (26) Google Scholar, 11Shetty 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 (41) Google Scholar, 12Bhandary 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 (62) Google Scholar, 13Bhandary 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 (66) Google Scholar indicating a causal link between increased p53 and AEC apoptosis in diverse forms of lung diseases. Recent reports also reveal that emphysema and PF concurrently occur in approximately 10% of smokers.23Morse D. Rosas I.O. Tobacco smoke-induced lung fibrosis and emphysema.Annu Rev Physiol. 2014; 76: 493-513Crossref PubMed Scopus (66) Google Scholar, 24Mitchell P.D. Das J.P. Murphy D.J. Keane M.P. Donnelly S.C. Dodd J.D. Butler M.W. Idiopathic pulmonary fibrosis with emphysema: evidence of synergy among emphysema and idiopathic pulmonary fibrosis in smokers.Respir Care. 2015; 60: 259-268Crossref PubMed Scopus (24) Google Scholar Similarly, patients who develop acute respiratory distress syndrome are more prone to the development of accelerated PF, which may likewise occur to some degree in up to 40% of patients.25Burnham E.L. Janssen W.J. Riches D.W.H. Moss M. Downey G.P. The fibroproliferative response in acute respiratory distress syndrome: mechanisms and clinical significance.Eur Respir J. 2014; 43: 276-285Crossref PubMed Scopus (203) Google Scholar, 26Martin C. Papazian L. Payan M.-J. Saux P. Gouin F. Pulmonary fibrosis correlates with outcome in adult respiratory distress syndrome: a study in mechanically ventilated patients.Chest. 1995; 107: 196-200Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Increased numbers of apoptotic AECs in the lungs of patients with IPF19Sutherland L.M. Edwards Y.S. Murray A.W. Alveolar type II cell apoptosis.Comp Biochem Physiol A Mol Integr Physiol. 2001; 129: 267-285Crossref PubMed Scopus (37) Google Scholar, 22Marudamuthu A.S. Shetty S.K. Bhandary Y.P. Karandashova S. Thompson M. Sathish V. Florova G. Hogan T.B. Pabelick C.M. Prakash Y.S. Tsukasaki Y. Fu J. Ikebe M. Idell S. Shetty S. Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.J Biol Chem. 2015; 290: 9428-9441Crossref PubMed Scopus (38) Google Scholar, 27Uhal B.D. Joshi I. Hughes W.F. Ramos C. Pardo A. Selman M. Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung.Am J Physiol Lung Cell Mol Physiol. 1998; 275: L1192-L1199PubMed Google Scholar or mice with established PF12Bhandary 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 (62) Google Scholar and protection against development of PF after transplantation of healthy AECs in mice with BLM28Serrano-Mollar A. Nacher M. Gay-Jordi G. Closa D. Xaubet A. Bulbena O. Intratracheal transplantation of alveolar type II cells reverses bleomycin-induced lung fibrosis.Am J Respir Crit Care Med. 2007; 176: 1261-1268Crossref PubMed Scopus (134) Google Scholar or silica-induced29Spitalieri P. Quitadamo M.C. Orlandi A. Guerra L. Giardina E. Casavola V. Novelli G. Saltini C. Sangiuolo F. Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells.Eur Respir J. 2012; 39: 446-457Crossref PubMed Scopus (44) Google Scholar lung injury underlines the importance of AEC apoptosis in fibrogenic remodeling. Furthermore, resistance to BLM or silica-induced AEC apoptosis and PF because of deficiency in p53 or PAI-1 expression12Bhandary 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 (62) Google Scholar, 14Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Fu J. Pinson B.M. Levin J. Shetty S. Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.Toxicol Appl Pharmacol. 2015; 283: 92-98Crossref PubMed Scopus (21) Google Scholar or inhibition of BLM-induced p53 from binding to endogenous urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and PAI-1 mRNAs13Bhandary 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 (66) Google Scholar demonstrates the contribution of increased p53 expression in AECs to the pathogenesis of lung injury and PF. Besides transcriptional activation and repression control of protein-coding genes, p53 also regulates miRNA encoding genes.30Feng Z. Zhang C. Wu R. Hu W. Tumor suppressor p53 meets microRNAs.J Mol Cell Biol. 2011; 3: 44-50Crossref PubMed Scopus (178) Google Scholar, 31Hermeking H. p53 Enters the microRNA world.Cancer Cell. 2007; 12: 414-418Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar miRNAs are 18 to 25 nucleotide long noncoding RNAs, which regulate pathophysiological functions via expression of target genes at the posttranscriptional level. The process involves degradation, destabilization,32Felekkis K. Touvana E. Stefanou C. Deltas C. microRNAs: a newly described class of encoded molecules that play a role in health and disease.Hippokratia. 2010; 14: 236-240PubMed Google Scholar or repression of translation,33Wilczynska A. Bushell M. The complexity of miRNA-mediated repression.Cell Death Differ. 2015; 22: 22-33Crossref PubMed Scopus (316) Google Scholar depending on the degree of homology of the miRNA with the target mRNA. Despite the fact that miRNAs are evolutionarily conserved, a single miRNA can target thousands of mRNAs, resulting in a lack of clarity about their true function or place in pathophysiologic events, including those that are central to the development of PF. Among these miRNAs, miR-34a appears to show marked induction by p53.34Raver-Shapira N. Marciano E. Meiri E. Spector Y. Rosenfeld N. Moskovits N. Bentwich Z. Oren M. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis.Mol Cell. 2007; 26: 731-743Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar We inferred that miR-34a may promote p53-mediated apoptosis, senescence, cell cycle arrest, and inhibition of proliferation.35Fridman J.S. Lowe S.W. Control of apoptosis by p53.Oncogene. 2003; 22: 9030-9040Crossref PubMed Scopus (1147) Google Scholar Interestingly, miR-34a inhibits deacetylation of p53 by blocking Sirt1 expression by binding to sequences present in the 3′ untranslated region of Sirt1 mRNA leading to increased acetylation of p53.36Solomon J.M. Pasupuleti R. Xu L. McDonagh T. Curtis R. DiStefano P.S. Huber L.J. Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage.Mol Cell Biol. 2006; 26: 28-38Crossref PubMed Scopus (392) Google Scholar These observations suggest that miR-34a–mediated acetylation of p53 could be a key effector of AEC apoptosis and development of PF through increased expression of p53 and by p53-mediated downstream changes in uPA fibrinolytic system cross talk. Currently available treatments are unable to target these interactions and prevent fibrogenesis. Therefore, we elucidated how p53 and miR-34a expression regulates AEC apoptosis and the development of PF through interaction with the major components of the uPA fibrinolytic system. Using isolated AECs from mice with BLM, silica, CS, and sepsis-induced lung injury, and a range of genetically altered mice subjected to BLM-induced lung injury, we further show how targeting this pathway mitigates AEC apoptosis and prevents PF. Wild-type (WT) and p53- and PAI-1–deficient mice of C57BL-6 background were either purchased from The Jackson Laboratory (Bar Harbor, ME) or bred at the University of Texas Health Science Center at Tyler. Breeding pairs of transgenic mice carrying SP-CCre and mTmG genes (SP-CCre/mTmG mice)37Kanegai C.M. Xi Y. Donne M.L. Gotts J.E. Driver I.H. Amidzic G. Lechner A.J. Jones K.D. Vaughan A.E. Chapman H.A. Rock J.R. Persistent pathology in influenza-infected mouse lungs.Am J Respir Cell Mol Biol. 2016; 55: 613-615Crossref PubMed Scopus (46) Google Scholar were kindly provided by Dr. Hal Chapman (University of California, San Francisco, CA). Transgenic uPA-deficient, or homozygous miR-34afl/fl and SP-CCre/mTmG mice of C57BL-6 lineage were bred at the University of Texas Health Science Center at Tyler. All animal experiments were approved by the University of Texas Health Science Center at Tyler Institutional Animal Care and Use Committee. AECs were isolated from the lungs of mice and the purity of AEC preparations was confirmed by lithium carbonate staining and immunoblotting for surfactant proteins A, B, and C (SP-A, SP-B, and SP-C, respectively), as described previously (90% to 95%).20Marudamuthu A.S. Bhandary Y.P. Shetty S.K. Fu J. Sathish V. Prakash Y. Shetty S. Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury.Am J Pathol. 2015; 185: 55-68Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar AECs were treated with 40 μg/mL BLM in the presence or absence of 10 μmol/L of caveolin-1 scaffolding domain peptide (CSP) or control scrambled peptide (CP) for 24 hours in AEC culture medium at 37°C, as we described.20Marudamuthu A.S. Bhandary Y.P. Shetty S.K. Fu J. Sathish V. Prakash Y. Shetty S. Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury.Am J Pathol. 2015; 185: 55-68Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The lysates and conditioned media were immunoblotted for changes in protein expression. AECs were isolated from deidentified histologically normal lungs of control subjects and IPF donor lungs, as previously described.20Marudamuthu A.S. Bhandary Y.P. Shetty S.K. Fu J. Sathish V. Prakash Y. Shetty S. Role of the urokinase-fibrinolytic system in epithelial-mesenchymal transition during lung injury.Am J Pathol. 2015; 185: 55-68Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Marudamuthu A.S. Shetty S.K. Bhandary Y.P. Karandashova S. Thompson M. Sathish V. Florova G. Hogan T.B. Pabelick C.M. Prakash Y.S. Tsukasaki Y. Fu J. Ikebe M. Idell S. Shetty S. Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.J Biol Chem. 2015; 290: 9428-9441Crossref PubMed Scopus (38) Google Scholar The purities of isolated cell preparations were confirmed, as described above, before immunoblotting. AEC lysates were subjected to Western blotting. In a separate experiment, human AECs purchased from ScienCell Res Lab (Carlsbad, CA) or isolated from normal lungs of control subjects were treated with BLM in the presence or absence of CSP or CP for 24 hours in AEC culture medium. The lysates and conditioned media were subjected to Western blotting. Mice were exposed to BLM or silica by intranasal instillation, as described earlier,12Bhandary 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 (62) Google Scholar, 13Bhandary 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 (66) Google Scholar, 14Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Fu J. Pinson B.M. Levin J. Shetty S. Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.Toxicol Appl Pharmacol. 2015; 283: 92-98Crossref PubMed Scopus (21) Google Scholar whereas those exposed to saline were used as controls. In a separate experiment, WT mice were subjected to cecal ligation puncture (CLP) to induce septic lung injury and AECs isolated 0 to 5 days later, as described.38Gao R. Ma Z. Hu Y. Chen J. Shetty S. Fu J. Sirt1 restrains lung inflammasome activation in a murine model of sepsis.Am J Physiol Lung Cell Mol Physiol. 2015; 308: L847-L853Crossref PubMed Scopus (85) Google Scholar In selected experiments, sham-operated mice were used as controls. Twenty-four hours after initiation of BLM, silica- or CLP-induced acute lung injury, mice were i.p. injected with either CSP or CP or remained untreated, as described.10Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Midde K.K. Ji H.-L. Shams H. Subramaniam R. Fu J. Idell S. Shetty S. Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.PLoS One. 2015; 10: e0123187Crossref Scopus (26) Google Scholar, 12Bhandary 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 (62) Google Scholar, 14Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Fu J. Pinson B.M. Levin J. Shetty S. Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.Toxicol Appl Pharmacol. 2015; 283: 92-98Crossref PubMed Scopus (21) Google Scholar Three days later, mice were euthanized and AECs were isolated. For chronic lung injury, WT mice were exposed to passive CS from 40 research cigarettes over a 2-hour period 5 days/week for 5 days (≤90 mg/m3 total solid particulates) using a mechanical smoking chamber (Teague Enterprises, Davis, CA). Control mice were exposed to ambient air, as we reported elsewhere.10Bhandary Y.P. Shetty S.K. Marudamuthu A.S. Midde K.K. Ji H.-L. Shams H. Subramaniam R. Fu J. Idell S. Shetty S. Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.PLoS One. 2015; 10: e0123187Crossref Scopus (26) Google Scholar, 11Shetty 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 (41) Google Scholar After 4 weeks, mice exposed to CS were i.p. injected with CSP or CP once a week for 4 weeks and these mice were exposed to CS for a total of 20 weeks, as described earlier,11Shetty 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 (41) Google Scholar then euthanized and AECs were isolated. The cell lysates and RNA isolated from AECs of WT mice with BLM, silica, CS, or CLP-induced lung injury and treated with or without CSP or CP, as well as AECs of control mice were subjected to Western blotting or real-time PCR to assess changes in p15Sp53, Acp53, p53, Sirt1, or cleaved and total caspase-3 proteins or miR-34a expression, respectively. cDNA corresponding to miR-34a antisense (miR-As) or Precursor-miR-34a (Pre-miR) sequences were subcloned into a pSMPUW universal promoterless lentivirus (Lv) vector purchased from Cell Biolab Inc. (San Diego, CA) downstream of the human SP-B 5′flanking DNA (-911/41) linked to the Luciferase reporter gene, as we described earlier.13Bhandary 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 (66) Google Scholar 293T cells were obtained from ATCC (Manassas, VA) and maintained in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum. The Lv vector constructs were transfected into 293T cells to obtain phage particles and viral titers, measured as per the manufacturer's protocol. Because SP-B promoter is AEC and Clara cell specific in the lungs, these constructs were later used to express the miR-As or Pre-miR in AECs in vivo. Six- to eight-week-old (with approximate 20 g body weight) WT mice or p53-, uPA-, and PAI-1–deficient mice were transduced with or without Lv vector harboring SP-B promoter alone (Lv-Ev) or Lv having SP-B promoter expressing miR-As (Lv-miR-As) or Pre-m
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