Abstract
Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a serious clinical concern and myofibroblasts have been suggested to have a major role, with it recently being revealed that some of these myofibroblasts are derived from lung epithelial cells through epithelial–mesenchymal transition (EMT). In this study, we examined the EMT-inducing abilities of drugs known to induce ILD clinically. EMT-like phenotypes were induced by A771726, an active metabolite of leflunomide having an inhibitory effect on dihydroorotate dehydrogenase (DHODH). Smad-interacting protein 1 (a transcription factor regulating EMT) and the Notch-signaling pathway but not transforming growth factor-β was shown to be involved in A771726-induced EMT-like phenotypes. When the cultures were supplemented with exogenous uridine, the A771726-induced EMT-like phenotypes and activation of the Notch-signaling pathway disappeared. Similarly, an A771726 analog without inhibitory activity on DHODH produced no induction, suggesting that this process is mediated through the inhibition of DHODH. In vivo, administration of leflunomide stimulated bleomycin-induced EMT-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fibrosis, both of which were suppressed by coadministration of uridine. Taken together, these findings suggest that leflunomide-dependent exacerbation of bleomycin-induced pulmonary fibrosis is mediated by stimulation of EMT of lung epithelial cells, providing the first evidence that drug-induced pulmonary fibrosis involves EMT of these cells.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- 6-MP:
-
6-mercaptopurine
- α-SMA:
-
α-smooth muscle actin
- BSA:
-
bovine serum albumin
- CTGF:
-
connective tissue growth factor
- DAPI:
-
4,6-diamino-2-phenylindole
- DAPT:
-
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester
- DHODH:
-
dihydroorotate dehydrogenase
- DMBA:
-
4-(dimethylamino)-benzaldehyde
- Dll-1:
-
Delta-like 1
- ECM:
-
extracellular matrix
- EMT:
-
epithelial–mesenchymal transition
- FBS:
-
fetal bovine serum
- ILD:
-
interstitial lung disease
- MMP:
-
matrix metallopeptidase
- NICD:
-
Notch intracellular domain
- proSP-C:
-
precursor of surfactant protein-C
- ∣SIP-1:
-
Smad-interacting protein 1
- TIMP:
-
tissue inhibitor of matrix metalloprotease
- TGF:
-
transforming growth factor
References
Olson AL, Swigris JJ, Lezotte DC, Norris JM, Wilson CG, Brown KK . Mortality from pulmonary fibrosis increased in the United States from 1992 to 2003. Am J Respir Crit Care Med 2007; 176: 277–284.
Fumeaux T, Rothmeier C, Jolliet P . Outcome of mechanical ventilation for acute respiratory failure in patients with pulmonary fibrosis. Intensive Care Med 2001; 27: 1868–1874.
Camus P, Fanton A, Bonniaud P, Camus C, Foucher P . Interstitial lung disease induced by drugs and radiation. Respiration 2004; 71: 301–326.
Inoue A, Saijo Y, Maemondo M, Gomi K, Tokue Y, Kimura Y et al. Severe acute interstitial pneumonia and gefitinib. Lancet 2003; 361: 137–139.
Flieder DB, Travis WD . Pathologic characteristics of drug-induced lung disease. Clin Chest Med 2004; 25: 37–45.
Moore BB, Hogaboam CM . Murine models of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2008; 294: L152–L160.
Sheppard D . Transforming growth factor beta: a central modulator of pulmonary and airway inflammation and fibrosis. Proc Am Thorac Soc 2006; 3: 413–417.
Kinnula VL, Myllarniemi M . Oxidant-antioxidant imbalance as a potential contributor to the progression of human pulmonary fibrosis. Antioxid Redox Signal 2008; 10: 727–738.
Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML, Gabbiani G . The myofibroblast: one function, multiple origins. Am J Pathol 2007; 170: 1807–1816.
Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S et al. IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. J Clin Invest 2007; 117: 3786–3799.
Kisseleva T, Brenner DA . Fibrogenesis of parenchymal organs. Proc Am Thorac Soc 2008; 5: 338–342.
Willis BC, Borok Z . TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 2007; 293: L525–L534.
Kasai H, Allen JT, Mason RM, Kamimura T, Zhang Z . TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res 2005; 6: 56.
Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 2006; 103: 13180–13185.
Kim KK, Wei Y, Szekeres C, Kugler MC, Wolters PJ, Hill ML et al. Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest 2009; 119: 213–224.
Smolen JS, Kalden JR, Scott DL, Rozman B, Kvien TK, Larsen A et al. Efficacy and safety of leflunomide compared with placebo and sulphasalazine in active rheumatoid arthritis: a double-blind, randomised, multicentre trial. European Leflunomide Study Group. Lancet 1999; 353: 259–266.
Emery P, Breedveld FC, Lemmel EM, Kaltwasser JP, Dawes PT, Gomor B et al. A comparison of the efficacy and safety of leflunomide and methotrexate for the treatment of rheumatoid arthritis. Rheumatology (Oxford) 2000; 39: 655–665.
Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol 1999; 93: 198–208.
Scott DL . Interstitial lung disease and disease modifying anti-rheumatic drugs. Lancet 2004; 363: 1239–1240.
Sakai F, Noma S, Kurihara Y, Yamada H, Azuma A, Kudoh S et al. Leflunomide-related lung injury in patients with rheumatoid arthritis: imaging features. Mod Rheumatol 2005; 15: 173–179.
Mejlvang J, Kriajevska M, Vandewalle C, Chernova T, Sayan AE, Berx G et al. Direct repression of cyclin D1 by SIP1 attenuates cell cycle progression in cells undergoing an epithelial mesenchymal transition. Mol Biol Cell 2007; 18: 4615–4624.
Thiery JP, Sleeman JP . Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006; 7: 131–142.
Shirakihara T, Saitoh M, Miyazono K . Differential regulation of epithelial and mesenchymal markers by deltaEF1 proteins in epithelial mesenchymal transition induced by TGF-beta. Mol Biol Cell 2007; 18: 3533–3544.
Saito RA, Watabe T, Horiguchi K, Kohyama T, Saitoh M, Nagase T et al. Thyroid transcription factor-1 inhibits transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells. Cancer Res 2009; 69: 2783–2791.
Li JL, Harris AL . Notch signaling from tumor cells: a new mechanism of angiogenesis. Cancer Cell 2005; 8: 1–3.
Bray SJ . Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 2006; 7: 678–689.
Sahlgren C, Gustafsson MV, Jin S, Poellinger L, Lendahl U . Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proc Natl Acad Sci USA 2008; 105: 6392–6397.
Wang Z, Li Y, Kong D, Banerjee S, Ahmad A, Azmi AS et al. Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway. Cancer Res 2009; 69: 2400–2407.
Kuo EA, Hambleton PT, Kay DP, Evans PL, Matharu SS, Little E et al. Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl-3-hydroxy-N-[3′-methyl-4′-(trifluoromethyl)phenyl] propenamide and related compounds. J Med Chem 1996; 39: 4608–4621.
Hatse S, De Clercq E, Balzarini J . Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. Biochem Pharmacol 1999; 58: 539–555.
Yukawa E, Mori S, Ueda K, Nakada Y . Population pharmacokinetic investigation of low-dose methotrexate in rheumatoid arthritics Japanese patients. J Clin Pharm Ther 2007; 32: 573–578.
Chan V, Charles BG, Tett SE . Population pharmacokinetics and association between A77 1726 plasma concentrations and disease activity measures following administration of leflunomide to people with rheumatoid arthritis. Br J Clin Pharmacol 2005; 60: 257–264.
Baumann P, Mandl-Weber S, Volkl A, Adam C, Bumeder I, Oduncu F et al. Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells. Mol Cancer Ther 2009; 8: 366–375.
Tang X, Yang J, Li J . Accelerative effect of leflunomide on recovery from hepatic fibrosis involves TRAIL-mediated hepatic stellate cell apoptosis. Life Sci 2009; 84: 552–557.
Liu T, Hu B, Choi YY, Chung M, Ullenbruch M, Yu H et al. Notch1 signaling in FIZZ1 induction of myofibroblast differentiation. Am J Pathol 2009; 174: 1745–1755.
Hamilton LC, Vojnovic I, Warner TD . A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo-oxygenase-2 in vitro and in vivo in a substrate-sensitive manner. Br J Pharmacol 1999; 127: 1589–1596.
Xu X, Williams JW, Bremer EG, Finnegan A, Chong AS . Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem 1995; 270: 12398–12403.
Inokuma S, Sato T, Sagawa A, Matsuda T, Takemura T, Ohtsuka T et al. Proposals for leflunomide use to avoid lung injury in patients with rheumatoid arthritis. Mod Rheumatol 2008; 18: 442–446.
Isohama Y, Kanemaru M, Kai H, Takahama K, Miyata T . Interaction between beta-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells. Life Sci 2001; 68: 2361–2371.
Isohama Y, Matsuo T, Kai H, Takahama K, Miyata T . Changes in beta 1- and beta 2-adrenoceptor mRNA levels in alveolar type II cells during cultivation. Biochem Mol Biol Int 1995; 36: 561–568.
Namba T, Homan T, Nishimura T, Mima S, Hoshino T, Mizushima T . Up-regulation of S100P expression by non-steroidal anti-inflammatory drugs and its role in anti-tumorigenic effects. J Biol Chem 2009; 284: 4158–4167.
Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254.
Mima S, Tsutsumi S, Ushijima H, Takeda M, Fukuda I, Yokomizo K et al. Induction of claudin-4 by nonsteroidal anti-inflammatory drugs and its contribution to their chemopreventive effect. Cancer Res 2005; 65: 1868–1876.
Woessner Jr JF . The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961; 93: 440–447.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Edited by RA Knight
Supplementary Information accompanies the paper on Cell Death and Differentiation website
Rights and permissions
About this article
Cite this article
Namba, T., Tanaka, KI., Ito, Y. et al. Induction of EMT-like phenotypes by an active metabolite of leflunomide and its contribution to pulmonary fibrosis. Cell Death Differ 17, 1882–1895 (2010). https://doi.org/10.1038/cdd.2010.64
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2010.64
