Fibroblast Growth Factor 2 Augments Transforming Growth Factor Beta 1 Induced Epithelial-mesenchymal Transition in Lung Cell Culture Model
Impaired lung epithelial cell regeneration following injury may contribute to the development of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is a critical event in embryonic development, wound healing following injury, and even cancer progression. Previous studies have shown that the combination of transforming growth factor beta-1 (TGFβ1) and fibroblast growth factor 2 (FGF2) induces EMT during cancer metastasis. However, this synergy remains to be elucidated in inducing EMT associated with wound healing after injury. We set out this study to determine the effect of fibroblast growth factor 2 (FGF2) on TGFβ1-induced EMT in the human lung epithelium.
BEAS-2B and A549 cells were treated with TGFβ1, FGF2, or both. EMT phenotype was investigated morphologically and by measuring mRNA expression levels; using quantitative real-time PCR. E-cadherin expression was assayed by western blot and immunofluorescence staining. Cell migration was confirmed using a wound-healing assay.
TGFβ1 induced a morphological change and a significant increase in cell migration of BEAS-2B cells. TGFβ1 significantly reduced E-cadherin (CDH1) mRNA expression and markedly induced expression of N-cadherin (CDH2), tenascin C (TNC), fibronectin (FN), actin alpha 2 (ACTA2), and collagen I (COL1A1). While FGF2 alone did not significantly alter EMT gene expression, it enhanced TGFβ1-induced suppression of CDH1 and upregulation of ACTA2, but not TNC, FN, and CDH2. FGF2 significantly inhibited TGFβ1-induced COL1A1 expression. Furthermore, FGF2 maintained TGFβ1-induced morphologic changes and increased the migration of TGFβ1-treated cells.
This study suggests a synergistic effect between TGFβ1 and FGF2 in inducing EMT in lung epithelial cells, which may play an important role in wound healing and tissue repair after injury.
2. King TE, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet;378(9807):1949–61.
3. Yang J, Wheeler SE, Velikoff M, Kleaveland KR, Lafemina MJ, Frank JA, et al. Activated alveolar epithelial cells initiate fibrosis through secretion of mesenchymal proteins. Am J Pathol. 2013;183(5):1559–70.
4. Li M, Luan F, Zhao Y, Hao H, Zhou Y, Han W, et al. Epithelial-mesenchymal transition: An emerging target in tissue fibrosis. Exp Biol Med. 2016;241(1):1–13.
5. Rout-Pitt N, Farrow N, Parsons D, Donnelley M. Epithelial mesenchymal transition (EMT): A universal process in lung diseases with implications for cystic fibrosis pathophysiology. Respir Res. 2018;19(1):136.
6. Salton F, Volpe MC, Confalonieri M. Epithelial-mesenchymal transition in the pathogenesis of idiopathic pulmonary fibrosis. Medicina. 2019;55(4):83.
7. 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. 2006;103(35):13180–5.
8. Tanjore H, Xu XC, Polosukhin V V., Degryse AL, Li B, Han W, et al. Contribution of Epithelial-derived Fibroblasts to Bleomycin-induced Lung Fibrosis. Am J Respir Crit Care Med. 2009;180(7):657–65.
9. Degryse AL, Tanjore H, Xu XC, Polosukhin V V., Jones BR, McMahon FB, et al. Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis. Am J Physiol Cell Mol Physiol. 2010;299(4):L442–52.
10. Ward C, Forrest IA, Murphy DM, Johnson GE, Robertson H, Cawston TE, et al. Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients. Thorax. 2005;60(10):865–71.
11. Harada T, Nabeshima K, Hamasaki M, Uesugi N, Watanabe K, Iwasaki H. Epithelial-mesenchymal transition in human lungs with usual interstitial pneumonia: Quantitative immunohistochemistry. Pathol Int. 2010;60(1):14–21.
12. Wu Z, Yang L, Cai L, Zhang M, Cheng X, Yang X, et al. Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an α-smooth muscle actin-Cre transgenic mouse. Respir Res. 2007;8(1):1.
13. Rock JR, Barkauskas CE, Cronce MJ, Xue Y, Harris JR, Liang J, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci. 2011;108(52):E1475–83.
14. Hoyles RK, Derrett-Smith EC, Khan K, Shiwen X, Howat SL, Wells AU, et al. An Essential Role for Resident Fibroblasts in Experimental Lung Fibrosis Is Defined by Lineage-Specific Deletion of High-Affinity Type II Transforming Growth Factor β Receptor. Am J Respir Crit Care Med. 2011;183(2):249–61.
15. Yamada M, Kuwano K, Maeyama T, Hamada N, Yoshimi M, Nakanishi Y, et al. Dual-immunohistochemistry provides little evidence for epithelial–mesenchymal transition in pulmonary fibrosis. Histochem Cell Biol. 2008;129(4):453–62.
16. Kolahian S, Fernandez IE, Eickelberg O, Hartl D. Immune Mechanisms in Pulmonary Fibrosis. Am J Respir Cell Mol Biol [Internet]. 2016;55(3):309–22.
17. Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 2003;112(12):1776–84.
18. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial–mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172(7):973–81.
19. Kalluri R. EMT: When epithelial cells decide to become mesenchymal-like cells. J Clin Invest. 2009;119(6):1417–9.
20. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009;119(6):1429–37.
21. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420–8.
22. Tennakoon A, Izawa T, Kuwamura M, Yamate J. Pathogenesis of Type 2 Epithelial to Mesenchymal Transition (EMT) in Renal and Hepatic Fibrosis. J Clin Med. 2015;5(1):4.
23. Miettinen PJ, Ebner R, Lopez AR, Derynck R. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol. 1994;127(6 Pt 2):2021–36.
24. Fan J-M, Ng Y-Y, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, et al. Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int. 1999;56(4):1455–67.
25. Kaimori A, Potter J, Kaimori J-Y, Wang C, Mezey E, Koteish A. Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro. J Biol Chem. 2007;282(30):22089–101.
26. Zheng G, Lyons JG, Tan TK, Wang Y, Hsu T-T, Min D, et al. Disruption of E-Cadherin by Matrix Metalloproteinase Directly Mediates Epithelial-Mesenchymal Transition Downstream of Transforming Growth Factor-β1 in Renal Tubular Epithelial Cells. Am J Pathol. 2009;175(2):580–91.
27. Hales AM, Schulz MW, Chamberlain CG, McAvoy JW. TGF-beta 1 induces lens cells to accumulate alpha-smooth muscle actin, a marker for subcapsular cataracts. Curr Eye Res. 1994;13(12):885–90.
28. Saika S, Kono-Saika S, Ohnishi Y, Sato M, Muragaki Y, Ooshima A, et al. Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury. Am J Pathol. 2004;164(2):651–63.
29. Kasai H, Allen JT, Mason RM, Kamimura T, Zhang Z. TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res. 2005;6(1):56.
30. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, et al. 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(5):1321–32.
31. Doerner AM, Zuraw BL. TGF-beta1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1beta but not abrogated by corticosteroids. Respir Res. 2009;10(1):100.
32. Kamitani S, Yamauchi Y, Kawasaki S, Takami K, Takizawa H, Nagase T, et al. Simultaneous Stimulation with TGF-β1 and TNF-α Induces Epithelial Mesenchymal Transition in Bronchial Epithelial Cells. Int Arch Allergy Immunol. 2011;155(2):119–28.
33. Kim JH, Jang YS, Eom K-S, Hwang Y Il, Kang HR, Jang SH, et al. Transtorming Growth Factor β1 Induces Epithelial-to-Mesenchymal Transition of A549 Cells. J Korean Med Sci. 2007;22(5):898.
34. Shintani Y, Maeda M, Chaika N, Johnson KR, Wheelock MJ. Collagen I Promotes Epithelial-to-Mesenchymal Transition in Lung Cancer Cells via Transforming Growth Factor–β Signaling. Am J Respir Cell Mol Biol. 2008;38(1):95–104.
35. Câmara J, Jarai G. Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-α. Fibrogenesis Tissue Repair. 2010;3(1):2.
36. Chen X-F, Zhang H-J, Wang H-B, Zhu J, Zhou W-Y, Zhang H, et al. Transforming growth factor-β1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways. Mol Biol Rep. 2012;39(4):3549–56.
37. Kawata M, Koinuma D, Ogami T, Umezawa K, Iwata C, Watabe T, et al. TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells. J Biochem. 2012;151(2):205–16.
38. O’Beirne SL, Walsh SM, Fabre A, Reviriego C, Worrell JC, Counihan IP, et al. CXCL9 Regulates TGF-β1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells. J Immunol. 2015;195(6):2788–96.
39. Kobayashi K, Koyama K, Suzukawa M, Igarashi S, Hebisawa A, Nagase T, et al. Epithelial-mesenchymal transition promotes reactivity of human lung adenocarcinoma A549 cells to CpG ODN. Allergol Int. 2016;65:S45–52.
40. Hackett T-L, Warner SM, Stefanowicz D, Shaheen F, Pechkovsky D V, Murray LA, et al. Induction of Epithelial–Mesenchymal Transition in Primary Airway Epithelial Cells from Patients with Asthma by Transforming Growth Factor-β1. Am J Respir Crit Care Med. 2009;180(2):122–33.
41. Qi L, Song W, Li L, Cao L, Yu Y, Song C, et al. FGF4 induces epithelial-mesenchymal transition by inducing store-operated calcium entry in lung adenocarcinoma. Oncotarget. 2016;7(45):74015–30.
42. Strutz F, Zeisberg M, Ziyadeh FN, Yang C-Q, Kalluri R, Müller GA, et al. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int. 2002;61(5):1714–28.
43. Lau M-T, So W-K, Leung PCK. Fibroblast Growth Factor 2 Induces E-Cadherin Down-Regulation via PI3K/Akt/mTOR and MAPK/ERK Signaling in Ovarian Cancer Cells. Migliaccio A, editor. PLoS One. 2013;8(3):e59083.
44. Lee JG, Jung E, Heur M. Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium. J Biol Chem. 2018;293(10):3758–69.
45. Masola V, Gambaro G, Tibaldi E, Brunati AM, Gastaldello A, D’Angelo A, et al. Heparanase and syndecan-1 interplay orchestrates fibroblast growth factor-2-induced epithelial-mesenchymal transition in renal tubular cells. J Biol Chem. 2012;287(2):1478–88.
46. Shirakihara T, Horiguchi K, Miyazawa K, Ehata S, Shibata T, Morita I, et al. TGF-β regulates isoform switching of FGF receptors and epithelial-mesenchymal transition. EMBO J. 2011;30(4):783–95.
47. Chen J, Chen G, Yan Z, Guo Y, Yu M, Feng L, et al. TGF-β1 and FGF2 Stimulate the Epithelial-Mesenchymal Transition of HERS Cells Through a MEK-Dependent Mechanism. J Cell Physiol. 2014;229(11):1647–59.
48. Chen P-Y, Qin L, Li G, Tellides G, Simons M. Fibroblast growth factor (FGF) signaling regulates transforming growth factor beta (TGFβ)-dependent smooth muscle cell phenotype modulation. Sci Rep. 2016;6(1):33407.
49. Kurimoto R, Iwasawa S, Ebata T, Ishiwata T, Sekine I, Tada Y, et al. Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation. Int J Oncol. 2016;48(5):1825–36.
50. Kurimoto R, Ebata T, Iwasawa S, Ishiwata T, Tada Y, Tatsumi K, et al. Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma. Oncol Lett. 2017;14(1):944–50.
51. Gholami MD, Falak R, Heidari S, Khoshmirsafa M, Kazemi MH, Zarnani A-H, et al. A Truncated Snail1 Transcription Factor Alters the Expression of Essential EMT Markers and Suppresses Tumor Cell Migration in a Human Lung Cancer Cell Line. Recent Pat Anticancer Drug Discov. 2019;14(2):158–69.
52. Guzy RD, Stoilov I, Elton TJ, Mecham RP, Ornitz DM. Fibroblast Growth Factor 2 Is Required for Epithelial Recovery, but Not for Pulmonary Fibrosis, in Response to Bleomycin. Am J Respir Cell Mol Biol. 2015;52(1):116–28.
53. Koo HY, El-Baz LMF, House SL, Cilvik SN, Dorry SJ, Shoukry NM, et al. Fibroblast growth factor 2 decreases bleomycin-induced pulmonary fibrosis and inhibits fibroblast collagen production and myofibroblast differentiation. J Pathol. 2018;246(1):54–66.
54. White SR, Fischer BM, Marroquin BA, Stern R. Interleukin-1beta mediates human airway epithelial cell migration via NF-kappaB. Am J Physiol Lung Cell Mol Physiol. 2008;295(6):L1018-27.
55. Schelch K, Wagner C, Hager S, Pirker C, Siess K, Lang E, et al. FGF2 and EGF induce epithelial–mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal. Carcinogenesis. 2018;39(4):534–45.
56. Tanaka T, Saika S, Ohnishi Y, Ooshima A, McAvoy JW, Liu C-Y, et al. Fibroblast growth factor 2: roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury. Mol Vis. 2004;10:462–7.
57. Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, Van Kooten C, et al. TGF-β1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2). Kidney Int. 2001;59(2):579–92.
58. Bossé Y, Thompson C, Stankova J, Rola-Pleszczynski M. Fibroblast Growth Factor 2 and Transforming Growth Factor β1 Synergism in Human Bronchial Smooth Muscle Cell Proliferation. Am J Respir Cell Mol Biol. 2006;34(6):746–53.
59. Xiao L, Du Y, Shen Y, He Y, Zhao H, Li Z. TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway. Front Biosci. 2012;17:2667–74.
60. Li F, Zhu T, Yue Y, Zhu X, Wang J, Liang L. Preliminary mechanisms of regulating PD‑L1 expression in non‑small cell lung cancer during the EMT process. Oncol Rep. 2018;40(2):775–82.
61. Hosper NA, van den Berg PP, de Rond S, Popa ER, Wilmer MJ, Masereeuw R, et al. Epithelial-to-mesenchymal transition in fibrosis: Collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition. Exp Cell Res. 2013;319(19):3000–9.
62. Pardo OE, Latigo J, Jeffery RE, Nye E, Poulsom R, Spencer-Dene B, et al. The Fibroblast Growth Factor Receptor Inhibitor PD173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo. Cancer Res. 2009;69(22):8645–51.
|Issue||Vol 19 No 4 (2020)|
|Epithelial cells Epithelial-mesenchymal transition Fibroblast growth factor 2 Lung injury Transforming growth factor beta1|
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