Title: Participation of miR-200 in Pulmonary Fibrosis
Abstract: Excessive extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). Epithelial-mesenchymal transition, involving transition of alveolar epithelial cells (AECs) to pulmonary fibroblasts, appears to be an important contributory process to lung fibrosis. Although aberrant expression of microRNAs (miRs) is involved in a variety of pathophysiologic processes, the role of miRs in fibrotic lung diseases is less well understood. In the present study, we found that miR-200a, miR-200b, and miR-200c are significantly down-regulated in the lungs of mice with experimental lung fibrosis. Levels of miR-200a and miR-200c were reduced in the lungs of patients with IPF. miR-200 had greater expression in AECs than in lung fibroblasts, and AECs from mice with experimental pulmonary fibrosis had diminished expression of miR-200. We found that the miR-200 family members inhibit transforming growth factor-β1–induced epithelial-mesenchymal transition of AECs. miR-200 family members can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Indeed, the introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Thus, the miR-200 family members participate importantly in fibrotic lung diseases and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach in treating pulmonary fibrotic diseases. Excessive extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). Epithelial-mesenchymal transition, involving transition of alveolar epithelial cells (AECs) to pulmonary fibroblasts, appears to be an important contributory process to lung fibrosis. Although aberrant expression of microRNAs (miRs) is involved in a variety of pathophysiologic processes, the role of miRs in fibrotic lung diseases is less well understood. In the present study, we found that miR-200a, miR-200b, and miR-200c are significantly down-regulated in the lungs of mice with experimental lung fibrosis. Levels of miR-200a and miR-200c were reduced in the lungs of patients with IPF. miR-200 had greater expression in AECs than in lung fibroblasts, and AECs from mice with experimental pulmonary fibrosis had diminished expression of miR-200. We found that the miR-200 family members inhibit transforming growth factor-β1–induced epithelial-mesenchymal transition of AECs. miR-200 family members can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Indeed, the introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Thus, the miR-200 family members participate importantly in fibrotic lung diseases and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach in treating pulmonary fibrotic diseases. Fibroblast activation with generation of provisional extracellular matrix (ECM) is a primary tissue response to injury.1Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3198) Google Scholar Successful wound repair relies on a balance of ECM synthesis and resolution, as well as re-epithelization of damaged epithelial surfaces.1Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3198) Google Scholar, 2Wynn T.A. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases.J Clin Invest. 2007; 117: 524-529Crossref PubMed Scopus (1122) Google Scholar Abnormal tissue repair is often associated with excessive ECM production that ultimately leads to fibrosis, including idiopathic pulmonary fibrosis (IPF).1Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Myofibroblasts and mechano-regulation of connective tissue remodelling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3198) Google Scholar, 3Thannickal V.J. Toews G.B. White E.S. Lynch 3rd, J.P. Martinez F.J. Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (546) Google Scholar ECM-producing lung fibroblasts arise from several sources, including the following: i) resident pulmonary fibroblasts, ii) circulating fibrocytes that then infiltrate into the lung, and iii) alveolar epithelial cells (AECs) through a process termed epithelial-mesenchymal transition (EMT).4Hardie W.D. Glasser S.W. Hagood J.S. Emerging concepts in the pathogenesis of lung fibrosis.Am J Pathol. 2009; 175: 3-16Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5Chapman H.A. Epithelial-mesenchymal interactions in pulmonary fibrosis.Annu Rev Physiol. 2011; 73: 413-435Crossref PubMed Scopus (313) Google Scholar EMT is a biological process that allows an epithelial cell to undergo multiple biochemical changes, resulting in mesenchymal cell features, including enhanced migratory capacity, production of ECM components, and loss of epithelial cell characteristics.6Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7457) Google Scholar, 7Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7679) Google Scholar EMT has been an essential step during implantation of the fertilized ovum, embryogenesis, and organ development.6Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7457) Google Scholar, 7Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7679) Google Scholar However, it also appears to be an important source of fibroblasts during repair of tissue injury associated with pathological fibrotic processes.6Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7457) Google Scholar, 7Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7679) Google Scholar Transforming growth factor (TGF)-β1 is a central mediator of lung fibrosis and can induce EMT of AECs both in vitro and in vivo.8Yao H.W. Xie Q.M. Chen J.Q. Deng Y.M. Tang H.F. TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro.Life Sci. 2004; 76: 29-37Crossref PubMed Scopus (101) Google Scholar, 9Xu J. Lamouille S. Derynck R. TGF-beta-induced epithelial to mesenchymal transition.Cell Res. 2009; 19: 156-172Crossref PubMed Scopus (1985) Google Scholar, 10Willis B.C. Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L525-L534Crossref PubMed Scopus (867) Google Scholar, 11Willis B.C. Liebler J.M. Luby-Phelps K. Nicholson A.G. Crandall E.D. du Bois R.M. Borok Z. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis.Am J Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, 12Kasai H. Allen J.T. Mason R.M. Kamimura T. Zhang Z. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT).Respir Res. 2005; 6: 56Crossref PubMed Scopus (628) Google Scholar, 13Kim 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 (1032) Google Scholar, 14Wu Z. Yang L. Cai L. Zhang M. Cheng X. Yang X. Xu J. Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre transgenic mouse.Respir Res. 2007; 8: 1Crossref PubMed Scopus (122) Google Scholar, 15Degryse A.L. Tanjore H. Xu X.C. Polosukhin V.V. Jones B.R. Boomershine C.S. Ortiz C. Sherrill T.P. McMahon F.B. Gleaves L.A. Blackwell T.S. Lawson W.E. TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.Am J Physiol Lung Cell Mol Physiol. 2011; 300: L887-L897Crossref PubMed Scopus (60) Google Scholar The mechanism by which TGF-β1 induces EMT involves recruitment of several transcriptional repressors, including SNAI1, SNAI2, Twist1, ZEB1, and ZEB2, to the promoters of genes required to preserve epithelial cell phenotypes.9Xu J. Lamouille S. Derynck R. TGF-beta-induced epithelial to mesenchymal transition.Cell Res. 2009; 19: 156-172Crossref PubMed Scopus (1985) Google Scholar Furthermore, TGF-β1 induces differentiation of pulmonary fibroblasts into myofibroblasts that are characterized by expression of smooth muscle actin (SMA)-α and elevated synthesis of ECM proteins.16Cutroneo K.R. White S.L. Phan S.H. Ehrlich H.P. Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression.J Cell Physiol. 2007; 211: 585-589Crossref PubMed Scopus (145) Google Scholar, 17Lee C.G. Cho S. Homer R.J. Elias J.A. Genetic control of transforming growth factor-beta1-induced emphysema and fibrosis in the murine lung.Proc Am Thorac Soc. 2006; 3: 476-477Crossref PubMed Scopus (26) Google Scholar MicroRNAs (miRs) are noncoding small RNAs, 22 nucleotides in length, that bind to the 3′ UTR of target genes and, thereby, repress translation and/or induce degradation of target gene mRNAs.18Stefani G. Slack F.J. Small non-coding RNAs in animal development.Nat Rev Mol Cell Biol. 2008; 9: 219-230Crossref PubMed Scopus (1143) Google Scholar miRs have regulated numerous molecular and cellular processes.18Stefani G. Slack F.J. Small non-coding RNAs in animal development.Nat Rev Mol Cell Biol. 2008; 9: 219-230Crossref PubMed Scopus (1143) Google Scholar Aberrant expression of miRs is associated with initiation and progression of pathological processes, including diabetes, cancer, and cardiovascular disease.19Croce C.M. Causes and consequences of microRNA dysregulation in cancer.Nat Rev Genet. 2009; 10: 704-714Crossref PubMed Scopus (2600) Google Scholar, 20Latronico M.V. Condorelli G. MicroRNAs and cardiac pathology.Nat Rev Cardiol. 2009; 6: 419-429Crossref PubMed Scopus (256) Google Scholar, 21Pandey A.K. Agarwal P. Kaur K. Datta M. MicroRNAs in diabetes: tiny players in big disease.Cell Physiol Biochem. 2009; 23: 221-232Crossref PubMed Scopus (147) Google Scholar, 22Thum T. Gross C. Fiedler J. Fischer T. Kissler S. Bussen M. Galuppo P. Just S. Rottbauer W. Frantz S. Castoldi M. Soutschek J. Koteliansky V. Rosenwald A. Basson M.A. Licht J.D. Pena J.T. Rouhanifard S.H. Muckenthaler M.U. Tuschl T. Martin G.R. Bauersachs J. Engelhardt S. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.Nature. 2008; 456: 980-984Crossref PubMed Scopus (1966) Google Scholar However, the role of miRs in TGF-β1–induced EMT of AECs and in lung fibrosis is not well characterized.23Pandit K.V. Corcoran D. Yousef H. Yarlagadda M. Tzouvelekis A. Gibson K.F. Konishi K. Yousem S.A. Singh M. Handley D. Richards T. Selman M. Watkins S.C. Pardo A. Ben-Yehudah A. Bouros D. Eickelberg O. Ray P. Benos P.V. Kaminski N. Inhibition and role of let-7d in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2010; 182: 220-229Crossref PubMed Scopus (421) Google Scholar An improved understanding of the roles that specific miRs play in the pathogenesis of lung fibrosis is likely to suggest important new directions for the treatment of IPF and other interstitial lung diseases. In the present study, we demonstrate that three miR-200 family members, including miR-200a, miR-200b, and miR-200c, are significantly down-regulated in the lungs of mice with bleomycin-induced lung fibrosis. The levels of miR-200a and miR-200c were decreased in the lungs of patients with IPF. We found that the miR-200 family members inhibit TGF-β1–induced EMT of AECs and can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Furthermore, we found that introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Overall, these data suggest that miR-200 family members play an important role in the pathogenesis of lung fibrosis and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach for the treatment of pulmonary fibrotic diseases. The experimental pulmonary fibrosis model was established, as previously described.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar The animal protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. For miR-200 treatment, control mimics (1.5 mg/kg body weight in 40 μL of saline), control mimics plus bleomycin (1.5 mg/kg mimics plus 1 U/kg bleomycin in 40 μL of saline), miR-200c mimics (1.5 mg/kg in 40 μL of saline), or miR-200c mimics plus bleomycin (1.5 mg/kg mimics plus 1 U/kg bleomycin in 40 μL of saline) were instilled intratracheally through oropharyngeal cavities. At 14 days after the treatment, the severity of lung fibrosis was evaluated. Human recombinant TGF-β1 was obtained from Peprotech (Rocky Hill, NJ). miR mimics and inhibitors were from Ambion (Grand Island, NY). miR array analysis was performed as previously described.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar Briefly, total RNAs were isolated from mouse lungs harvested at days 0, 7, and 14 after bleomycin instillation (three mice per group) with the miRNAeasy Mini Kit (Qiagen, Valencia, CA). The miR array was performed using the miRCURY LNA microRNA Array (Exiqon, Woburn, MA). The miR profiling data were deposited into Array Express (accession number E-MEXP-2749) and are free to public access. Primary AECs were isolated from saline- and bleomycin-treated mice, as previously described, with modifications.25Konigshoff M. Kramer M. Balsara N. Wilhelm J. Amarie O.V. Jahn A. Rose F. Fink L. Seeger W. Schaefer L. Gunther 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 In brief, lungs were lavaged through the right ventricle with 10 mL of sterile PBS, and tissues were minced and digested with 0.1% collagenase, 0.005% trypsin, and 0.04% DNase. The suspension was filtered through 40-μm nylon meshes and centrifuged at 200 × g for 10 minutes. The pellet was resuspended in modified Eagle's media, and negative selection for lymphocytes/macrophages was performed by incubation on CD16/32- and CD45-coated Petri dishes for 30 minutes at 37°C. Negative selection for fibroblasts was performed by adherence for 45 minutes on cell culture dishes. The adherent lung fibroblasts from the previously described procedures were cultured in modified Eagle's media containing 10% fetal bovine serum (FBS). The fibroblasts at passage 2 were trypsinized, and the same numbers of cells were plated for experiments. AECs or lung fibroblasts from each mouse were used as an independent line. Four to five mice were used for each condition in the study. Isolation and culture of primary rat AECs were performed essentially as previously described.26Chen L. Bosworth C.A. Pico T. Collawn J.F. Varga K. Gao Z. Clancy J.P. Fortenberry J.A. Lancaster Jr, J.r. Matalon S. DETANO and nitrated lipids increase chloride secretion across lung airway cells.Am J Respir Cell Mol Biol. 2008; 39: 150-162Crossref PubMed Scopus (32) Google Scholar Before being treated with TGF-β1, the cells were starved in media containing 0.5% FBS for 24 hours. The human primary pulmonary fibroblast line, MRC-5, and the rat ATII cell line, RLE-6TN, were obtained from American Type Culture Collection (Manassas, VA) and cultured according to the manufacturer's instructions. IPF and histologically normal lung tissue samples were obtained from the NIH Lung Tissue Research Consortium and the University of Alabama at Birmingham Tissue Procurement and Cell Culture Core. The protocol was approved by the Institutional Review Board at the University of Alabama at Birmingham. The assay was performed as previously described.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar, 27Liu G. Friggeri A. Yang Y. Park Y.J. Tsuruta Y. Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses.Proc Natl Acad Sci U S A. 2009; 106: 15819-15824Crossref PubMed Scopus (360) Google Scholar TaqMan probes for miR-200a, miR-200b, miR-200c, RNU48, snoRNA, and sno135 were obtained from Applied Biosystems (Carlsbad, CA). The expression of SMA-α, fibronectin (Fn), collagen 1A1, E-cadherin, GATA3, fibroblast-specific protein (FSP) 1, zona occludens-1 (ZO-1), ZEB1, and ZEB2 was determined using the SYBR Green Master Mix kit (Roche, Indianapolis, IN). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sequences were as follows: human Fn, 5′-GTGTTGGGAATGGTCGTGGGGAATG-3′ (sense) and 5′-CCAATGCCACGGCCATAGCAGTAGC-3′ (antisense); mouse Fn, 5′-TCTGGGAAATGGAAAAGGGGAATGG-3′ (sense) and 5′-CACTGAAGCAGGTTTCCTCGGTTGT-3′ (antisense); mouse SMA-α, 5′-GACGCTGAAGTATCCGATAGAACACG-3′ (sense) and 5′-CACCATCTCCAGAGTCCAGCACAAT-3′ (antisense); mouse collagen 1A1, 5′-GGAGGGCGAGTGCTGTGCTTT-3′ (sense) and 5′-GGGACCAGGAGGACCAGGAAGT-3′ (antisense); rat ZEB1, 5′-TTTGTCTCCCAGTCAGCCACCTTTA-3′ (sense) and 5′-GGAATCTGTCCAGCTTGCATCTTTT-3′ (antisense); rat ZEB2, 5′-GCAGCACTTAGGTGTAGGGTTAGAAGC-3′ (sense) and 5′-GACCGACGGCTGGAATACTAGGAGA-3′ (antisense); rat GAPDH, 5′-ATGCTGGTGCTGAGTATGTCGTGGAG-3′ (sense) and 5′-TGAGGGAGTTGTCATATTTCTCGTGGTTC-3′ (antisense); mouse E-cadherin, 5′-GTGTGCTCACCTCTGGGCTGGAC-3′ (sense) and 5′-GAGTGTTGGGGGCATCATCATCG-3′ (antisense); mouse GAPDH, 5′-CGACTTCAACAGCAACTCCCACTCTTCC-3′ (sense) and 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′ (antisense); human GAPDH, 5′-GCTGGCGCTGAGTACGTCGTGGAGT-3′ (sense) and 5′-CACAGTCTTCTGGGTGGCAGTGATGG-3′ (antisense); mouse ZO-1, 5′-TCTGGCATCATTCGCCTTCATACA-3′ (sense) and 5′-CGCATAATTAAGACGATCAACCGC-3′ (antisense); mouse FSP1, 5′-TCCACAAATACTCAGGCAAAGAGGG-3′ (sense) and 5′-TGTTGCTGTCCAAGTTGCTCATCAC-3′ (antisense); and rat GATA3, 5′-CCATTACCACCTATCCGCCCTAT-3′ (sense) and 5′-GCAGTTCACACACTCCCTGCCTT-3′ (antisense). Western blot analysis was performed as previously described.28Liu G. Park Y.J. Abraham E. Interleukin-1 receptor-associated kinase (IRAK)-1-mediated NF-kappaB activation requires cytosolic and nuclear activity.FASEB J. 2008; 22: 2285-2296Crossref PubMed Scopus (54) Google Scholar Mouse anti-Fn antibody and rabbit anti-GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-SMA-α was from Sigma-Aldrich (St. Louis, MO). Mouse anti-vimentin antibody was from Abcam (Cambridge, MA). Mouse anti-E-cadherin and mouse anti-N-cadherin antibodies were from BD Bioscience (Sparks, MA). Immunofluorescence assays were performed as previously described.28Liu G. Park Y.J. Abraham E. Interleukin-1 receptor-associated kinase (IRAK)-1-mediated NF-kappaB activation requires cytosolic and nuclear activity.FASEB J. 2008; 22: 2285-2296Crossref PubMed Scopus (54) Google Scholar Confocal microscopy was also performed. Immunohistochemistry (IHC) assays were performed as previously described.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar Briefly, sections of mouse lungs (10 μm thick) were prepared, deparaffinized with xylene, and then rehydrated in water. Antigen retrieval was performed in a pressure cooker in Tris-EDTA solution, pH 9.0. After incubation in Tris-buffered saline with Tween for 10 minutes and 3% H2O2 for 10 minutes, the sections were blocked with avidin-biotin blocker and affinity-purified goat anti-mouse IgG [heavy chain (H) and light chain (L)]. The sections were then incubated with mouse anti-α-SMA (Sigma) overnight at 4°C, secondary antibody for 45 minutes, avidin–horseradish peroxidase for 45 minutes, and diaminobenzidine (DAB) chromogen, sequentially. Finally, the sections were counterstained with hematoxylin. The right lungs from mice were collected and homogenized in 5 mL of 0.5 mol/L acetic acid in PBS containing 0.6% pepsin. The extracts were rotated at 4°C overnight and cleared by centrifugation at 17,200 × g for 15 minutes. Collagen content was measured using the Sircol Collagen Assay kit (Biocolor Ltd, Carrickfergus, UK), according to the manufacturer's instructions. Collagen content is presented as micrograms of acid-soluble collagen per right lung. One-way analysis of variance, followed by the Holm-Sidak or Tukey-Kramer test, was performed for multiple group comparisons. The Student's t-test was used for comparison between two groups. P < 0.05 was considered significant. To examine alterations of miR in pulmonary fibrosis, we performed miR array analyses on RNA samples isolated from lungs of mice that were given intratracheal bleomycin, a well-studied pulmonary fibrosis model.29Chua F. Gauldie J. Laurent G.J. Pulmonary fibrosis: searching for model answers.Am J Respir Cell Mol Biol. 2005; 33: 9-13Crossref PubMed Scopus (228) Google Scholar, 30Moore B.B. Hogaboam C.M. Murine models of pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L152-L160Crossref PubMed Scopus (602) Google Scholar We found that several miRs demonstrate altered expression in fibrotic mouse lungs.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar We found that miR-21, an miR with enhanced expression in fibrotic mouse lungs, regulates pulmonary fibrogenesis in response to bleomycin-induced lung injury.24Liu G. Friggeri A. Yang Y. Milosevic J. Ding Q. Thannickal V.J. Kaminski N. Abraham E. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis.J Exp Med. 2010; 207: 1589-1597Crossref PubMed Scopus (756) Google Scholar In our effort to characterize miRs with reduced expression in fibrotic mouse lungs, we defined the role of miR-200 family members in the regulation of pulmonary fibrosis. To confirm the alterations of miR-200 expression in fibrotic lungs, we performed real-time PCR analysis and found that the expression of miR-200 family members, including miR-200a, miR-200b, and miR-200c, is time dependently decreased in murine lungs after intratracheal injection of bleomycin (Figure 1). These data suggest that miR-200 may play a role in the regulation of lung fibrosis. The AECs and lung fibroblasts are the most relevant cell populations involved in pulmonary fibrosis. To determine the levels of miR-200 in these two cell populations, we isolated AECs and lung fibroblasts from control untreated mice and from mice after bleomycin administration. We found that the expression of miR-200 is down-regulated in the AECs isolated from mice with experimental lung fibrosis (Figure 2A). Furthermore, we found that miR-200 is expressed in the AECs at remarkably higher levels than in the lung fibroblasts (Figure 2B). These data suggest that the diminished levels of miR-200 in the fibrotic lungs are primarily derived from the decrease in the expression of miR-200 in AECs. The AECs demonstrated much greater expression of E-cadherin and ZO-1 (two epithelial cell markers), whereas they demonstrated decreased amounts of Fn and FSP1 (two fibroblast markers), than did lung fibroblasts. These data indicate the purity of the studied cell populations (Figure 2C). Because AECs undergoing EMT are a major source of lung fibroblasts that produce excessive ECM in fibrotic lungs, the diminished expression of miR-200 in AECs suggests that miR-200 may participate in the regulation of EMT involving AECs during lung fibrosis. To determine whether the expression of miR-200 family members is altered in the lungs of patients with IPF, we measured the levels of miR-200 in control normal human lung tissue and in the lungs of patients with IPF. As shown in Figure 3A, the levels of miR-200a and miR-200c were significantly decreased in the lungs of patients with IPF compared with those in the healthy controls, although miR-200b demonstrated a slight increase in the lungs of patients with IPF. The expression of two ECM proteins, collagen 1A1 and Fn, was increased in the lungs of patients with IPF (Figure 3B), consistent with previous studies.25Konigshoff M. Kramer M. Balsara N. Wilhelm J. Amarie O.V. Jahn A. Rose F. Fink L. Seeger W. Schaefer L. Gunther 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, 31Wang X.M. Zhang Y. Kim H.P. Zhou Z. Feghali-Bostwick C.A. Liu F. Ifedigbo E. Xu X. Oury T.D. Kaminski N. Choi A.M. Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis.J Exp Med. 2006; 203: 2895-2906Crossref PubMed Scopus (324) Google Scholar TGF-β1 is one of the major mediators of pulmonary fibrosis and induces EMT of AECs in vitro.8Yao H.W. Xie Q.M. Chen J.Q. Deng Y.M. Tang H.F. TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro.Life Sci. 2004; 76: 29-37Crossref PubMed Scopus (101) Google Scholar, 9Xu J. Lamouille S. Derynck R. TGF-beta-induced epithelial to mesenchymal transition.Cell Res. 2009; 19: 156-172Crossref PubMed Scopus (1985) Google Scholar, 10Willis B.C. Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L525-L534Crossref PubMed Scopus (867) Google Scholar, 11Willis B.C. Liebler J.M. Luby-Phelps K. Nicholson A.G. Crandall E.D. du Bois R.M. Borok Z. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis.Am J Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, 12Kasai H. Allen J.T. Mason R.M. Kamimura T. Zhang Z. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT).Respir Res. 2005; 6: 56Crossref PubMed Scopus (628) Google Scholar, 13Kim 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 (1032) Google Scholar, 14Wu Z. Yang L. Cai L. Zhang M. Cheng X. Yang X. Xu J. Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre transgenic mouse.Respir Res. 2007; 8: 1Crossref PubMed Scopus (122) Google Scholar, 15Degryse A.L. Tanjore H. Xu X.C. Polosukhin V.V. Jones B.R. Boomershine C.S. Ortiz C. Sherrill T.P. McMahon F.B. Gleaves L.A. Blackwell T.S. Lawson W.E. TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.Am J Physiol Lung Cell Mol Physiol. 2011; 300: L887-L897Crossref PubMed Scopus (60) Google Scholar To investigate whether miR-200 has a role in TGF-β1–induced EMT transition, we first sought to determine whether TGF-β1 regulates miR-200 expression in AECs. In these experiments, the rat alveolar type II cell line, RLE-6TN, was treated with TGF-β1 for 0 to 3 days. As shown in Figure 4A, the expression of miR-200a, miR-200b, and miR-200c was significantly decreased after TGF-β1 treatment. These findings suggest that miR-200 may play a role in TGF-β1–induced EMT of AECs. To exclude the possibility that the diminished expression of miR-200 after TGF-β1 treatment only occurs in immortalized AEC lines, we treated rat primary alveolar type II cells with TGF-β1. As shown in Figure 4B, TGF-β1 treatment markedly diminished miR-200 expression in the primary rat alveolar type II cells. The EMT process is characterized by the loss of epithelial cell phenotypes, such as diminished expression of E-cadherin and ZO-1, and the acquisition of mesenchymal cell phenotypes, such as enhanced expression of ECM proteins, vimentin, SMA-α, and N-cadherin.6Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7457) Google Scholar, 7Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7679) Google Scholar To study the role of miR-200 in the EMT of AECs during pulmonary fibrosis, we first determined the expression of E-cadherin in the lungs of bleomycin- treated mice. As shown in Supplemental Figure S1A (available at http://ajp.amjpathol.org), the expression of E-cadherin in the lungs of mice with bleomycin-induced pulmonary fibrosis is markedly decreased compared with that in normal control lungs. More specifically, we found that the expression of E-cadherin is significantly decreased in AECs isolated from mice with bleomycin-induced lung fibrosis (see Supplemental Figure S1B at http://ajp.amjpathol.org). In addition, the expression of Fn was significantly increased in AECs isolated from mice with experimental lung fibrosis (see Supplemental Figure S1B at http://ajp.amjpathol.org). These data confirm previous findings8Yao H.W. Xie Q.M. Chen J.Q. Deng Y.M. Tang H.F. TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro.Life Sci. 2004; 76: 29-37Crossref PubMed Scopus (101) Google Scholar, 9Xu J. Lamouille S. Derynck R. 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To determine the role of miR-200 in modulating EMT, we transfected RLE-6TN cells with control miR mimics or mimics for miR-200a, miR-200b, or miR-200c and then treated the cells with TGF-β1. As shown in Figure 5, A and C, overexpression of miR-200, particularly miR-200b and miR-200c, attenuated TGF-β1–induced expression of the mesenchymal cell markers, including SMA-α and vimentin, and TGF-β1–induced mesenchymal cell morphological characteristics. Furthermore, we found that miR-200 enhanced E-cadherin expression and attenuated TGF-β1–repressed E-cadherin expression (Figure 5B). Inhibition of miR-200 enhanced TGF-β1–induced expression of mesenchymal markers, including SMA-α, vimentin, and N-cadherin in rat AECs (Figure 6), although such effects were less striking, as suggested by those found with miR-200 mimics. This may be because of relatively low basal levels of miR-200s in the rat cell line. Together, these data suggest that miR-200 has an inhibitory role in TGF-β1–induced EMT in AECs.Figure 6Inhibition of miR-200 enhances EMT in AECs. RLE-6TN cells were transfected with 40 nmol/L control (con) miR inhibitors or inhibitors to miR-200a, miR-200b, or miR-200c. Three days after the transfection, the cells were starved in media containing 0.5% FBS for 24 hours and then treated with 5 ng/mL TGF-β1 for 48 hours. The cells were collected, and cell extracts were prepared. The levels of vimentin, SMA-α, N-cadherin, and the loading control, actin, were determined by using Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) miRs regulate various cellular processes by down-regulating the expression of their target genes. As shown in Supplemental Figure S2 (available at http://ajp.amjpathol.org), transfection of miR-200 mimics significantly decreased the mRNA levels of GATA3, ZEB1, and ZEB2, transcriptional factors that have regulated EMT,32Yan W. Cao Q.J. Arenas R.B. Bentley B. Shao R. GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition.J Biol Chem. 2010; 285: 14042-14051Crossref PubMed Scopus (162) Google Scholar, 33Gregory P.A. Bert A.G. Paterson E.L. Barry S.C. Tsykin A. Farshid G. Vadas M.A. Khew-Goodall Y. Goodall G.J. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.Nat Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3197) Google Scholar in rat AECs. These data suggest that the ability of miR-200 to diminish expression of GATA3, ZEB1, and ZEB2 may be a mechanism by which miR-200 inhibits EMT in AECs. Lung fibroblasts function as effectors in pulmonary fibrosis by producing excessive collagen and Fn.4Hardie W.D. Glasser S.W. Hagood J.S. Emerging concepts in the pathogenesis of lung fibrosis.Am J Pathol. 2009; 175: 3-16Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar The finding that miR-200 inhibits EMT in AECs suggests that restoration of expression of miR-200 family members in the lungs may have potential therapeutic utility in the treatment of pulmonary fibrosis. However, although we have shown that miR-200 is primarily expressed in AECs, miR-200 mimics may be taken up by both AECs and lung fibroblasts if they are introduced intratracheally or systemically. To determine whether miR-200 has a role in regulating the fibrogenic activity of lung fibroblasts, we transfected primary human lung fibroblasts with control or miR-200 mimics. As shown in Figure 7, overexpression of miR-200, particularly miR-200b and miR-200c, markedly attenuated TGF-β1–induced expression of Fn and SMA-α. Given that TGF-β1 is a central mediator of lung fibrosis, these data suggest that therapies able to increase miR-200 expression in the lungs may be able to diminish TGF-β1–mediated fibrogenesis and may attenuate lung fibrosis. Lung fibroblasts can differentiate into myofibroblasts that are characterized by enhanced expression of SMA-α and elevated fibrogenic activity.4Hardie W.D. Glasser S.W. Hagood J.S. Emerging concepts in the pathogenesis of lung fibrosis.Am J Pathol. 2009; 175: 3-16Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar To determine whether miR-200 participates in the regulation of this process, we isolated lung fibroblasts from control mice or those with experimental pulmonary fibrosis. As shown in Figure 8A, lung fibroblasts from mice with experimental pulmonary fibrosis demonstrated significantly enhanced expression of Fn and SMA-α compared with those from the lungs of untreated mice. These data are consistent with previous findings and suggest differentiation of fibroblasts into myofibroblasts in fibrotic lungs.25Konigshoff M. Kramer M. Balsara N. Wilhelm J. Amarie O.V. Jahn A. Rose F. Fink L. Seeger W. Schaefer L. Gunther 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, 34Ramos C. Montano M. Garcia-Alvarez J. Ruiz V. Uhal B.D. Selman M. Pardo A. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression.Am J Respir Cell Mol Biol. 2001; 24: 591-598Crossref PubMed Scopus (316) Google Scholar, 35Xia H. Diebold D. Nho R. Perlman D. Kleidon J. Kahm J. Avdulov S. Peterson M. Nerva J. Bitterman P. Henke C. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (177) Google Scholar Next, we transfected control miR or miR-200 mimics into lung fibroblasts from mice with experimental pulmonary fibrosis. We found that overexpression of miR-200, particularly miR-200b and miR-200c, significantly diminished the expression of SMA-α (Figure 8B), suggesting that miR-200 can reverse the differentiation of lung fibroblasts into myofibroblasts. To further define the role of miR-200 in regulating the fibrogenic activity of lung fibroblasts, we transfected control miR and miR-200 mimics into lung fibroblasts isolated from a patient with IPF and found that miR-200, particularly miR-200b and miR-200c, diminished the expression of SMA-α and Fn in these cells (Figure 8C). Taken together, our data suggest that introduction of miR-200 into lungs diminishes EMT in AECs, thereby reducing the numbers of ECM-producing lung fibroblasts, and also attenuates the fibrogenic activity of lung fibroblasts and reverses the differentiation of lung fibroblasts into myofibroblasts.