Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
권호 표지
DOI QR코드

DOI QR Code

Role of integrin α2 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells

  • Kawami, Masashi (Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University) ;
  • Ojima, Takamichi (Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University) ;
  • Yumoto, Ryoko (Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University) ;
  • Takano, Mikihisa (Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University)
  • Received : 2021.09.16
  • Accepted : 2022.02.23
  • Published : 2022.10.15
⟨ Previous Next ⟩

Abstract

Methotrexate (MTX) is widely used to treat various diseases. However, it induces adverse reactions like serious lung injury, including pulmonary fibrosis. Increasing evidence suggests that epithelial-mesenchymal transition (EMT) in injured alveolar epithelium contributes to the development of the pathophysiological state of the lung. We demonstrated that MTX induced EMT in cultured alveolar epithelial cell lines. Integrin-mediated signaling is considered a significant factor in recognizing the EMT process. However, the relationship between MTX-induced EMT and integrin family members is poorly understood. In the present study, we aimed to clarify the role of integrin in MTX-induced EMT in A549 and NCI-H1299 (H1299) cells by focusing on the integrin alpha 2 (ITGA2) subunit, selected based on our microarray analysis. MTX treatment for 72 h significantly increased the mRNA and cell surface expression of ITGA2 in both cell lines. However, this upregulation by MTX was suppressed by co-treatment with SB431542 and folic acid, which are inhibitors of MTX-induced EMT in A549 cells. The mRNA expression levels of EMT-related genes were more affected in the MTX-treated A549 cells with high ITGA2 expression than in those with low ITGA2 expression. Finally, E7820, an ITGA2 inhibitor, suppressed MTX-induced EMT-related phenotypic changes, such as morphology and mRNA and protein expression of α-smooth muscle actin, a representative EMT marker. These findings suggest that ITGA2 may play a key role in MTX-induced EMT in alveolar epithelial cells.

Keywords

Acknowledgement

This work was supported by the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JP18H02586, JP18K06749, and JP19K16447).

References

  1. Kozminski P, Halik PK, Chesori R, Gniazdowska E (2020) Overview of dual-acting drug methotrexate in different neurological diseases, autoimmune pathologies and cancers. Int J Mol Sci 14:3483. https://doi.org/10.3390/ijms2 11034 83
  2. Cronstein BN (2005) Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacol Rev 57:163-172. https://doi.org/10.1124/pr.57.2.3
  3. Chou RC, Kane M, Ghimire S, Gautam S, Gui J (2016) Treatment for rheumatoid arthritis and risk of alzheimer's disease: a nested case-control analysis. CNS Drugs 30:1111-1120. https://doi.org/10.1007/s40263-016-0374-z
  4. Zhou M, Xu R, Kaelber DC, Gurney ME (2020) Tumor Necrosis Factor (TNF) blocking agents are associated with lower risk for Alzheimer's disease in patients with rheumatoid arthritis and psoriasis. PLoS ONE 15:e0229819. https://doi.org/10.1371/journal.pone.0229819
  5. Karadag AS, Kanbay A, Ozlu E, Uzuncakmak TK, Gedik C, Akdeniz N (2015) Pulmonary fibrosis developed secondary to methotrexate use in a patient with psoriasis vulgaris. North Clin Istanbul 2:159-161. https://doi.org/10.14744/nci.2015.97759
  6. Jakubovic BD, Donovan A, Webster PM, Shear NH (2013) Methotrexate-induced pulmonary toxicity. Can Respir J 20:153-155. https://doi.org/10.1155/2013/527912
  7. Imokawa S, Colby TV, Leslie KO, Helmers RA (2000) Methotrexate pneumonitis: review of the literature and histopathological findings in nine patients. Eur Respir J 15:373-381. https://doi.org/10.1034/j.1399-3003.2000.15b25.x
  8. Nieto MA, Huang RY, Jackson RA, Thiery JP (2016) Emt: 2016. Cell 166:21-45. https://doi.org/10.1016/j.cell.2016.06.028
  9. Chen T, You Y, Jiang H, Wang ZZ (2017) Epithelial-mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis. J Cell Physiol 232:3261-3272. https://doi.org/10.1002/jcp.25797
  10. Zhang Y, Weinberg RA (2018) Epithelial-to-mesenchymal transition in cancer: complexity and opportunities. Front Med 12:361-373. https://doi.org/10.1007/s11684-018-0656-6
  11. Stone RC, Pastar I, Ojeh N, Chen V, Liu S, Garzon KI, Tomic-Canic M (2016) Epithelial-mesenchymal transition in tissue repair and fibrosis. Cell Tissue Res 365:495-506. https://doi.org/10.1007/s00441-016-2464-0
  12. Salton F, Volpe MC, Confalonieri M (2019) Epithelial-mesenchymal transition in the pathogenesis of idiopathic pulmonary fibrosis. Medicina (Kaunas) 55:83. https://doi.org/10.3390/medicina55040083
  13. Bartis D, Mise N, Mahida RY, Eickelberg O, Thickett DR (2014) Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important? Thorax 69:760-765. https://doi.org/10.1136/thora xjnl-2013-204608
  14. Kawami M, Harabayashi R, Miyamoto M, Harada R, Yumoto R, Takano M (2016) Methotrexate-induced epithelial-mesenchymal transition in the alveolar epithelial cell line A549. Lung 194:923-930. https://doi.org/10.1007/s00408-016-9935-7
  15. Weng J, Chen H, Wu H, Tu M, Wang Z, Chen D, Wang Z, Chen C (2020) Amiodarone induces epithelial-mesenchymal transition in A549 cells via activation of TGF-β1. Drug Chem Toxicol 43:415-422. https://doi.org/10.1080/01480 545. 2018. 14970 55
  16. Weng CM, Li Q, Chen KJ, Xu CX, Deng MS, Li T, Zhang DD, Duan ZX, Chen ZQ, Li GH, Chen J, Wang JM (2020) Bleomycin induces epithelial-to-mesenchymal transition via bFGF/PI3K/ESRP1 signaling in pulmonary fibrosis. Biosci Rep. https://doi.org/10.1042/bsr20190756
  17. Tsukui T, Ueha S, Abe J, Hashimoto S, Shichino S, Shimaoka T, Shand FH, Arakawa Y, Oshima K, Hattori M, Inagaki Y, Tomura M, Matsushima K (2013) Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis. Am J Pathol 183:758-773. https://doi.org/10.1016/j.ajpath.2013.06.005
  18. Konaka T, Kawami M, Yamamoto A, Yumoto R, Takano M (2020) miR-484: a possible indicator of drug-induced pulmonary fibrosis. J Pharm Pharm Sci 23:486-495 https://doi.org/10.18433/jpps31448
  19. Kawami M, Takenaka S, Kadekaru Y, Akai M, Konaka T, Yumoto R, Takano M (2021) Evaluation on epithelial-mesenchymal state and microRNAs focusing on isolated alveolar epithelial cells from bleomycin injured rat lung. Toxicology 461:152903. https://doi.org/10.1016/j.tox.2021.152903
  20. Kawami M, Harabayashi R, Harada R, Yamagami Y, Yumoto R, Takano M (2018) Folic acid prevents methotrexate-induced epithelial-mesenchymal transition via suppression of secreted factors from the human alveolar epithelial cell line A549. Biochem Biophys Res Commun 497:457-463. https://doi.org/10.1016/j.bbrc.2018.02.111
  21. Yamagami Y, Kawami M, Ojima T, Futatsugi S, Yumoto R, Takano M (2020) Role of plasminogen activator inhibitor-1 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells. Biochem Biophys Res Commun 525:543-548. https://doi.org/10.1016/j.bbrc.2020.02.131
  22. Cohen P, Cross D, Janne PA (2021) Kinase drug discovery 20 years after imatinib: progress and future directions. Nat Rev Drug Discov 20:551-569. https://doi.org/10.1038/s41573-021-00195-4
  23. Kawami M, Harada R, Ojima T, Yamagami Y, Yumoto R, Takano M (2019) Association of cell cycle arrest with anticancer druginduced epithelial-mesenchymal transition in alveolar epithelial cells. Toxicology 424:152231. https://doi.org/10.1016/j.tox.2019.06.002
  24. Semba T, Funahashi Y, Ono N, Yamamoto Y, Sugi NH, Asada M, Yoshimatsu K, Wakabayashi T (2004) An angiogenesis inhibitor E7820 shows broad-spectrum tumor growth inhibition in a xenograft model: possible value of integrin alpha2 on platelets as a biological marker. Clin Cancer Res 10:1430-1438. https://doi.org/10.1158/1078-0432.ccr-0109-03
  25. Ojima T, Kawami M, Yumoto R, Takano M (2020) Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells. Toxicol Res 37:293-300. https://doi.org/10.1007/s43188-020-00067-w
  26. Kawami M, Honda N, Hara T, Yumoto R, Takano M (2019) Investigation on inhibitory effect of folic acid on methotrexate-induced epithelial-mesenchymal transition focusing on dihydrofolate reductase. Drug Metab Pharmacokinet 34:396-399. https://doi.org/10.1016/j.dmpk.2019.08.003
  27. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673-687. https://doi.org/10.1016/S0092-8674(02)00971-6
  28. Kechagia JZ, Ivaska J, Roca-Cusachs P (2019) Integrins as biomechanical sensors of the microenvironment. Nat Rev Mol Cell Biol 20:457-473. https://doi.org/10.1038/s41580-019-0134-2
  29. Adorno-Cruz V, Liu H (2019) Regulation and functions of integrin α2 in cell adhesion and disease. Genes Dis 6:16-24. https://doi.org/10.1016/j.gendis.2018.12.003
  30. Koenig A, Mueller C, Hasel C, Adler G, Menke A (2006) Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells. Cancer Res 66:4662-4671. https://doi.org/10.1158/0008-5472.can-05-2804
  31. Naci D, Vuori K, Aoudjit F (2015) Alpha2beta1 integrin in cancer development and chemoresistance. Semin Cancer Biol 35:145-153. https://doi.org/10.1016/j.semca ncer.2015.08.004
  32. Keizer RJ, Funahashi Y, Semba T, Wanders J, Beijnen JH, Schellens JH, Huitema AD (2011) Evaluation of α2-integrin expression as a biomarker for tumor growth inhibition for the investigational integrin inhibitor E7820 in preclinical and clinical studies. AAPS J 13:230-239. https://doi.org/10.1208/s12248-011-9260-2
  33. Mita M, Kelly KR, Mita A, Ricart AD, Romero O, Tolcher A, Hook L, Okereke C, Krivelevich I, Rossignol DP, Giles FJ, Rowinsky EK, Takimoto C (2011) Phase I study of E7820, an oral inhibitor of integrin alpha-2 expression with antiangiogenic properties, in patients with advanced malignancies. Clin Cancer Res 17:193-200. https://doi.org/10.1158/1078-0432.ccr-10-0010
  34. Uehara T, Minoshima Y, Sagane K, Sugi NH, Mitsuhashi KO, Yamamoto N, Kamiyama H, Takahashi K, Kotake Y, Uesugi M, Yokoi A, Inoue A, Yoshida T, Mabuchi M, Tanaka A, Owa T (2017) Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat Chem Biol 13:675-680. https://doi.org/10.1038/nchembio.2363
  35. Cheli Y, Kanaji S, Jacquelin B, Chang M, Nugent DJ, Kunicki TJ (2007) Transcriptional and epigenetic regulation of the integrin collagen receptor locus ITGA1-PELO-ITGA2. Biochim Biophys Acta 1769:546-558. https://doi.org/10.1016/j.bbaexp.2007.06.004
  36. Dong J, Wang R, Ren G, Li X, Wang J, Sun Y, Liang J, Nie Y, Wu K, Feng B, Shang Y, Fan D (2017) HMGA2-FOXL2 Axis regulates metastases and epithelial-to-mesenchymal transition of chemoresistant gastric cancer. Clin Cancer Res 23:3461-3473. https://doi.org/10.1158/1078-0432.ccr-16-2180
  37. Su YJ, Lin WH, Chang YW, Wei KC, Liang CL, Chen SC, Lee JL (2015) Polarized cell migration induces cancer type-specific CD133/integrin/Src/Akt/GSK3β/β-catenin signaling required for maintenance of cancer stem cell properties. Oncotarget 6:38029-38045. https://doi.org/10.18632/oncotarget.5703
  38. Wang Q, Cao T, Guo K, Zhou Y, Liu H, Pan Y, Hou Q, Nie Y, Fan D, Lu Y, Zhao X (2020) Regulation of integrin subunit alpha 2 by miR-135b-5p modulates chemoresistance in gastric cancer. Front Oncol 10:308. https://doi.org/10.3389/fonc.2020.00308
  39. Georgiou KR, Nadhanan RR, Fan CM, Xian CJ (2015) Methotrexate-induced bone marrow adiposity is mitigated by folinic acid supplementation through the regulation of Wnt/β-catenin signalling. J Cell Physiol 230:648-656. https://doi.org/10.1002/jcp.24788
  40. Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B (2007) Functional atlas of the integrin adhesome. Nat Cell Biol 9:858-867. https://doi.org/10.1038/ncb0807-858
  41. Aguirre Ghiso JA (2002) Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo. Oncogene 21:2513-2524. https://doi.org/10.1038/sj.onc.1205342
  42. Yamamoto A, Kawami M, Konaka T, Takenaka S, Yumoto R, Takano M (2019) Anticancer drug-induced epithelial-mesenchymal transition via p53/miR-34a axis in A549/ABCA3 cells. J Pharm Pharm Sci 22:516-524. https://doi.org/10.18433/jpps30660