Volume 12, Issue 2 (Vol.12 No.2 Jul 2023)                   rbmb.net 2023, 12(2): 211-219 | Back to browse issues page

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Karetnikova E S, Jarzebska N, Rodionov R N, Rubets E, Markov A G, Spieth* P M. mRNA Levels of Epithelial and Mesenchymal Markers in Lung Epithelial Cell Lines. rbmb.net 2023; 12 (2) :211-219
URL: http://rbmb.net/article-1-1176-en.html
Department of General Physiology, Saint-Petersburg State University, Saint-Petersburg, Russia.
Abstract:   (1152 Views)
Background: Epithelial-mesenchymal transition (EMT) is an important physiologic process that determines the outcome of lung tissue healing after injury. Stimuli and molecular cascades inducing EMT lead to up-regulation of the mesenchymal-specific genes in the alveolar epithelial cells and to down-regulation of the genes coding for epithelial markers. Alveolar epithelial cell lines are commonly used as in vitro models to study processes occurring in the lung tissue. The aim of this study is to quantify and compare mRNA expression levels of epithelial and mesenchymal markers in a number of lung epithelial cell lines.

Methods: Lung epithelial cell lines L2, R3/1 and RLE-6TN were cultured. Repeated mRNA isolation, reverse transcription, and quantitative PCR with primers to epithelial (E-cadherin, occludin, and ZO-2) and mesenchymal (α-SMA, collagen III, and vimentin) markers were performed.

Results: First, our study revealed a higher level of epithelial transcripts in the RLE-6TN cell line compared to L2 and R3/1 cells. Secondly, we have found simultaneous mRNA expression of both epithelial (E-cadherin, occludin and ZO-2) and mesenchymal (α-SMA, collagen III and vimentin) markers in all cell lines studied.

Conclusions: Our data indicate that at the transcriptional level the L2, R3/1, and RLE-6TN cell lines are at one of the intermediate stages of EMT, which opens new possibilities for the study of EMT on cell lines. Determination of the direction of changes in epithelial and mesenchymal markers will make it possible to establish the factors responsible for both EMT and reverse mesenchymal-epithelial transition.
Full-Text [PDF 676 kb]   (620 Downloads)    
Type of Article: Original Article | Subject: Cell Biology
Received: 2023/05/6 | Accepted: 2023/07/14 | Published: 2023/12/20

1. Stone RC, Pastar I, Ojeh N, Chen V, Liu S, Garzon KI, et al. Epithelial-mesenchymal transition in tissue repair and fibrosis. Cell Tissue Res. 2016;365(3):495-506. [DOI:10.1007/s00441-016-2464-0] [PMID] []
2. Bartis D, Mise N, Mahida RY, Eickelberg O, Thickett DR. Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important? Thorax. 2014;69(8):760-5. [DOI:10.1136/thoraxjnl-2013-204608] [PMID]
3. Jolly MK, Ward C, Eapen MS, Myers S, Hallgren O, Levine H, et al. Epithelial-mesenchymal transition, a spectrum of states: Role in lung development, homeostasis, and disease. Dev Dyn. 2018;247(3):346-58. [DOI:10.1002/dvdy.24541] [PMID]
4. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420-8. [DOI:10.1172/JCI39104] [PMID] []
5. 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(35):13180-5. [DOI:10.1073/pnas.0605669103] [PMID] []
6. Coopman P, Djiane A. Adherens Junction and E-Cadherin complex regulation by epithelial polarity. Cell Mol Life Sci. 2016;73(18):3535-53. [DOI:10.1007/s00018-016-2260-8] [PMID]
7. van Roy F, Berx G. The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci. 2008;65(23):3756-88. [DOI:10.1007/s00018-008-8281-1] [PMID]
8. Markov AG, Aschenbach JR, Amasheh S. Claudin clusters as determinants of epithelial barrier function. IUBMB Life. 2015;67(1):29-35. [DOI:10.1002/iub.1347] [PMID]
9. 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. [DOI:10.1186/s12931-018-0834-8] [PMID] []
10. Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer. 2016;15:18. [DOI:10.1186/s12943-016-0502-x] [PMID] []
11. Gunzel D, Fromm M. Claudins and other tight junction proteins. Compr Physiol. 2012;2(3):1819-52. [DOI:10.1002/cphy.c110045] [PMID]
12. Van Itallie CM, Anderson JM. Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol. 2014;36:157-65. [DOI:10.1016/j.semcdb.2014.08.011] [PMID] []
13. Ohta H, Chiba S, Ebina M, Furuse M, Nukiwa T. Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012;302(2):L193-205. [DOI:10.1152/ajplung.00349.2010] [PMID]
14. Yang L, Chen X, Simet SM, Hu G, Cai Y, Niu F, et al. Reactive Oxygen Species/Hypoxia-Inducible Factor-1alpha/Platelet-Derived Growth Factor-BB Autocrine Loop Contributes
15. to Cocaine-Mediated Alveolar Epithelial Barrier Damage. Am J Respir Cell Mol Biol. 2016;55(5):736-48. [DOI:10.1165/rcmb.2016-0096OC] [PMID] []
16. Cabrera-Benitez NE, Parotto M, Post M, Han B, Spieth PM, Cheng WE, et al. Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition. Crit Care Med. 2012;40(2):510-7. [DOI:10.1097/CCM.0b013e31822f09d7] [PMID] []
17. Nagaraja SS, Nagarajan D. Radiation-Induced Pulmonary Epithelial-Mesenchymal Transition: A Review on Targeting Molecular Pathways and Mediators. Curr Drug Targets. 2018;19(10):1191-204. [DOI:10.2174/1389450119666180207092234] [PMID]
18. Salton F, Volpe MC, Confalonieri M. Epithelial(-)Mesenchymal Transition in the Pathogenesis of Idiopathic Pulmonary Fibrosis. Medicina (Kaunas). 2019;55(4). [DOI:10.3390/medicina55040083] [PMID] []
19. Checa M, Hagood JS, Velazquez-Cruz R, Ruiz V, Garcia-De-Alba C, Rangel-Escareno C, et al. Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells. PLoS One. 2016;11(3):e0150383. [DOI:10.1371/journal.pone.0150383] [PMID] []
20. Zou D, Li J, Fan Q, Zheng X, Deng J, Wang S. Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury. J Cell Biochem. 2019;120(4):4837-50. [DOI:10.1002/jcb.27382] [PMID]
21. Chapman HA. Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol. 2011;73:413-35. [DOI:10.1146/annurev-physiol-012110-142225] [PMID]
22. Guarino M, Tosoni A, Nebuloni M. Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition. Hum Pathol. 2009;40(10):1365-76. [DOI:10.1016/j.humpath.2009.02.020] [PMID]
23. Willis BC, Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol. 2007;293(3):L525-34. [DOI:10.1152/ajplung.00163.2007] [PMID]
24. 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. [DOI:10.1016/S0002-9440(10)62351-6] [PMID]
25. Mahmoudi Rad M, Mahmoudi Rad N, Mirdamadi Y. Expression of TGF-beta3 in isolated fibroblasts from foreskin. Rep Biochem Mol Biol. 2015;3(2):76-81.
26. Chen H, Wu FP, Yang YZ, Yu XY, Zhang L, Zhang H, et al. Cigarette smoke extract induces the epithelial-to-mesenchymal transition via the PLTP/TGF-beta1/Smad2 pathway in RLE-6TN cells. Toxicol Res (Camb). 2017;6(2):215-22. [DOI:10.1039/C6TX00378H] [PMID] []
27. Ding Q, Subramanian I, Luckhardt TR, Che P, Waghray M, Zhao XK, et al. Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-beta. Am J Physiol Lung Cell Mol Physiol. 2017;312(6):L926-L35. [DOI:10.1152/ajplung.00121.2016] [PMID] []
28. Pullamsetti SS, Savai R, Dumitrascu R, Dahal BK, Wilhelm J, Konigshoff M, et al. The role of dimethylarginine dimethylaminohydrolase in idiopathic pulmonary fibrosis. Sci Transl Med. 2011;3(87):87ra53. [DOI:10.1126/scitranslmed.3001725] [PMID]
29. Makia R, Al-Sammarrae K, Al-Halbosiy M, Al-Mashhadani M. In Vitro Cytotoxic Activity of Total Flavonoid from Equisetum Arvense Extract. Rep Biochem Mol Biol. 2022;11(3):487-92. [DOI:10.52547/rbmb.11.3.487] [PMID] []
30. Su SD, Cong SG, Bi YK, Gao DD. Paraquat promotes the epithelial-mesenchymal transition in alveolar epithelial cells through regulating the Wnt/beta-catenin signal pathway. Eur Rev Med Pharmacol Sci. 2018;22(3):802-9.
31. Xu GP, Li QQ, Cao XX, Chen Q, Zhao ZH, Diao ZQ, et al. The effect of TGF-beta1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells. Cell Mol Biol Lett. 2007;12(3):457-72. [DOI:10.2478/s11658-007-0018-x] [PMID] []
32. Koslowski R, Barth K, Augstein A, Tschernig T, Bargsten G, Aufderheide M, et al. A new rat type I-like alveolar epithelial cell line R3/1: bleomycin effects on caveolin expression. Histochem Cell Biol. 2004;121(6):509-19. [DOI:10.1007/s00418-004-0662-4] [PMID]
33. Douglas WH, Kaighn ME. Clonal isolation of differentiated rat lung cells. In Vitro. 1974;10(3-4):230-7. [DOI:10.1007/BF02615237] [PMID]
34. Knebel JW, Aufderheide M, Emura M. Comparison of biological effects of different polycyclic aromatic hydrocarbons in lung cells of hamster and rat in vitro. Toxicol Lett. 1994;72(1-3):65-72. [DOI:10.1016/0378-4274(94)90011-6] [PMID]
35. Driscoll KE, Carter JM, Iype PT, Kumari HL, Crosby LL, Aardema MJ, et al. Establishment of immortalized alveolar type II epithelial cell lines from adult rats. In Vitro Cell Dev Biol Anim. 1995;31(7):516-27. [DOI:10.1007/BF02634029] [PMID]
36. Rentzsch I, Santos CL, Huhle R, Ferreira JMC, Koch T, Schnabel C, et al. Variable stretch reduces the pro-inflammatory response of alveolar epithelial cells. PLoS One. 2017;12(8):e0182369. [DOI:10.1371/journal.pone.0182369] [PMID] []
37. Barth K, Reh J, Sturrock A, Kasper M. Epithelial vs myofibroblast differentiation in immortal rat lung cell lines--modulating effects of bleomycin. Histochem Cell Biol. 2005;124(6):453-64. [DOI:10.1007/s00418-005-0048-2] [PMID]
38. Barth K, Blasche R, Kasper M. Lack of evidence for caveolin-1 and CD147 interaction before and after bleomycin-induced lung injury. Histochem Cell Biol. 2006;126(5):563-73. [DOI:10.1007/s00418-006-0192-3] [PMID]
39. Takano M, Yamamoto C, Yamaguchi K, Kawami M, Yumoto R. Analysis of TGF-beta1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3. Drug Metab Pharmacokinet. 2015;30(1):111-8. [DOI:10.1016/j.dmpk.2014.10.007] [PMID]
40. Horalkova L, Radziwon A, Endter S, Andersen R, Koslowski R, Radomski MW, et al. Characterisation of the R3/1 cell line as an alveolar epithelial cell model for drug disposition studies. Eur J Pharm Sci. 2009;36(4-5):444-50. [DOI:10.1016/j.ejps.2008.11.010] [PMID]
41. Balli D, Ustiyan V, Zhang Y, Wang IC, Masino AJ, Ren X, et al. Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J. 2013;32(2):231-44. [DOI:10.1038/emboj.2012.336] [PMID] []
42. Tanjore H, Xu XC, Polosukhin VV, 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. [DOI:10.1164/rccm.200903-0322OC] [PMID] []
43. Kawamoto M, Matsunami T, Ertl RF, Fukuda Y, Ogawa M, Spurzem JR, et al. Selective migration of alpha-smooth muscle actin-positive myofibroblasts toward fibronectin in the Boyden's blindwell chamber. Clin Sci (Lond). 1997;93(4):355-62. [DOI:10.1042/cs0930355] [PMID]
44. Felton VM, Borok Z, Willis BC. N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition. Am J Physiol Lung Cell Mol Physiol. 2009;297(5):L805-12. [DOI:10.1152/ajplung.00009.2009] [PMID] []
45. Li LC, Li DL, Xu L, Mo XT, Cui WH, Zhao P, et al. High-Mobility Group Box 1 Mediates Epithelial-to-Mesenchymal Transition in Pulmonary Fibrosis Involving Transforming Growth Factor-beta1/Smad2/3 Signaling. J Pharmacol Exp Ther. 2015;354(3):302-9. [DOI:10.1124/jpet.114.222372] [PMID]
46. Liang J, Zhang Y, Xie T, Liu N, Chen H, Geng Y, et al. Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice. Nat Med. 2016;22(11):1285-93. [DOI:10.1038/nm.4192] [PMID] []
47. Nagarajan D, Melo T, Deng Z, Almeida C, Zhao W. ERK/GSK3beta/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition. Free Radic Biol Med. 2012;52(6):983-92. [DOI:10.1016/j.freeradbiomed.2011.11.024] [PMID] []
48. Ramos C, Becerril C, Montano M, Garcia-De-Alba C, Ramirez R, Checa M, et al. FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway. Am J Physiol Lung Cell Mol Physiol. 2010;299(2):L222-31. [DOI:10.1152/ajplung.00070.2010] [PMID]

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