The effects of ezrin on transforming growth factor beta mediated epithelial mesenchymal transition_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

JIA Man , YAN Xiaoyi , JIANG Jie , MENG Yaqi , XU Jiayan ,  YAO Xin

Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangshu 210029, P.R.China;

Corresponding author：

YAO Xin, Email: yaoxin@njmu.edu.cn

Keywords：

Human bronchial epithelial cells; Epithelial mesenchymal transition; Transforming growth factor-β; Ezrin

DOI：

10.7507/1671-6205.201702015

Video：

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ObjectiveTo investigate the effects of ezrin on transforming growth factor beta (TGF-β) mediated epithelial mesenchymal transition (EMT). MethodsHuman bronchial epithelial 16HBE cells were stimulated by TGF-β to induce EMT. The mRNA and protein levels were analyzed by RT-PCR and Western blot. Lentivirus (LV)-Ezrin-shRNA was employed to investigate the effects of ezrin deficiency on cell morphology and expressions of EMT associated biomarkers including E-cadherin, vimentin, and alpha smooth muscle actin (α-SMA). The effects of recombinant ezrin protein on 16HBE were also examined. ResultsThe expression of ezrin was down-regulated in 16HBE activated by TGF-β. Due to ezrin depletion, the cell morphology changed from a typical multilateral paving stone-like appearance to a mesenchymal-like fusiform appearance along with the decreased expression of epithelium biomarker E-cadherin and the increased mesenchymal cell markers, vimentin and α-SMA. Recombination protein ezrin increased the expression of E-cadherin whilst reducing vimentin and α-SMA. ConclusionTGF-β promotes EMT of 16HBE cells at least partly via inhibiting the expression of ezrin.

Citation： JIA Man, YAN Xiaoyi, JIANG Jie, MENG Yaqi, XU Jiayan, YAO Xin. The effects of ezrin on transforming growth factor beta mediated epithelial mesenchymal transition. Chinese Journal of Respiratory and Critical Care Medicine, 2017, 16(3): 280-286. doi: 10.7507/1671-6205.201702015 Copy

1.	Bousquet J, Jeffery PK, Busse WW, et al. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med, 2000, 161(5): 1720-1745.
2.	Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease. Cell, 2009, 139(5): 871-890.
3.	Lee JM, Dedhar S, Kalluri R, et al. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol, 2006, 172(7): 973-981.
4.	Sohal SS, Walters EH. Role of epithelial mesenchymal transition (EMT) in chronic obstructive pulmonary disease (COPD). Respir Res, 2013, 14: 120.
5.	Nowrin K, Sohal SS, Peterson G, et al. Epithelial-mesenchymal transition as a fundamental underlying pathogenic process in COPD airways: fibrosis, remodeling and cancer. Expert Rev Respir Med, 2014, 8(5): 547-559.
6.	Heijink IH, Postma DS, Noordhoek JA, et al. House dust mite-promoted epithelial-to-mesenchymal transition in human bronchial epithelium. Am J Respir Cell Mol Biol, 2010, 42(1): 69-79.
7.	Haynes J, Srivastava J, Madson N, et al. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression. Mol Biol Cell, 2011, 22(24): 4750-4764.
8.	Fehon RG, McClatchey AI, Bretscher A. Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol, 2010, 11(4): 276-287.
9.	Moleirinho S, Tilston-Lunel A, Angus L, et al. The expanding family of FERM proteins. Biochem J, 2013, 452(2): 183-193.
10.	Valderrama F, Thevapala S, Ridley AJ. Radixin regulates cell migration and cell-cell adhesion through Rac1. J Cell Sci, 2012, 125(Pt 14): 3310-3319.
11.	Chiappetta C, Leopizzi M, Censi F, et al. Correlation of the Rac1/RhoA pathway with ezrin expression in osteosarcoma. Appl Immunohistochem Mol Morphol, 2014, 22(3): 162-170.
12.	Arumugam P, Partelli S, Coleman SJ, et al. Ezrin expression is an independent prognostic factor in gastro-intestinal cancers. J Gastrointest Surg, 2013, 17(12): 2082-2091.
13.	Kong J, Li Y, Liu S, et al. High expression of ezrin predicts poor prognosis in uterine cervical cancer. BMC Cancer, 2013, 13: 520.
14.	Leiphrakpam PD, Rajput A, Mathiesen M, et al. Ezrin expression and cell survival regulation in colorectal cancer. Cell Signal, 2014, 26(5): 868-879.
15.	Ren L, Hong SH, Cassavaugh J, et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene, 2009, 28(6): 792-802.
16.	Bretscher A. Purification of an 80, 000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J Cell Biol, 1983, 97(2): 425-432.
17.	Xie JJ, Zhang FR, Tao LH, et al. Expression of ezrin in human embryonic, fetal, and normal adult tissues. J Histochem Cytochem, 2011, 59(11): 1001-1008.
18.	Bonilha VL. Focus on molecules: ezrin. Exp Eye Res, 2007, 84(4): 613-614.
19.	Berryman M, Franck Z, Bretscher A. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci, 1993, 105(Pt 4): 1025-1043.
20.	Mangeat P, Roy C, Martin M. ERM proteins in cell adhesion and membrane dynamics. Trends Cell Biol, 1999, 9(5): 187-192.
21.	Di Cristofano C, Leopizzi M, Miraglia A, et al. Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell. Mod Pathol, 2010, 23(7): 1012-1020.
22.	Sarrió D, Rodríguez-Pinilla SM, Dotor A, et al. Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast Cancer Res Treat, 2006, 98(1): 71-79.
23.	Tokunou M, Niki T, Saitoh Y, et al. Altered expression of the ERM proteins in lung adenocarcinoma. Lab Invest, 2000, 80(11): 1643-1650.
24.	Chaw SY, Majeed AA, Dalley AJ, et al. Epithelial to mesenchymal transition (EMT) biomarkers--E-cadherin, beta-catenin, APC and Vimentin--in oral squamous cell carcinogenesis and transformation. Oral Oncol, 2012, 48(10): 997-1006.
25.	Araki K, Ebata T, Guo AK, et al. p53 regulates cytoskeleton remodeling to suppress tumor progression. Cell Mol Life Sci, 2015, 72(21): 4077-4094.
26.	McVicker CG, Leung SY, Kanabar V, et al. Repeated allergen inhalation induces cytoskeletal remodeling in smooth muscle from rat bronchioles. Am J Respir Cell Mol Biol, 2007, 36(6): 721-727.
27.	Sahai E, Marshall CJ. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol, 2003, 5(8): 711-719.
28.	Aubert JD, Dalal BI, Bai TR, et al. Transforming growth factor β1 gene expression in human airways. Thorax, 1994, 49(3): 225-232.
29.	Zavadil J, Böttinger EP. TGF-β and epithelial-to-mesenchymal transitions. Oncogene, 2005, 24(37): 5764-5774.
30.	Vega G, Alarcón S, San Martín R. The cellular and signalling alterations conducted by TGF-β contributing to renal fibrosis. Cytokine, 2016, 88: 115-125.
31.	O'Connor JW, Riley PN, Nalluri SM, et al. Matrix rigidity mediates TGFβ1-induced epithelial-myofibroblast transition by controlling cytoskeletal organization and MRTF-A localization. J Cell Physiol, 2015, 230(8): 1829-1839.
32.	Chen MJ, Gao XJ, Xu LN, et al. Ezrin is required for epithelial-mesenchymal transition induced by TGF-β 1 in A549 cells. Int J Oncol, 2014, 45(4): 1515-1522.
33.	Mani SA, Guo W, Liao MJ, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell, 2008, 133(4): 704-715.
34.	Phanish MK, Wahab NA, Colville-Nash P, et al. The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells. Biochem J, 2006, 393(Pt 2): 601-607.
35.	Zhang YE. Non-Smad signaling pathways of the TGF-β family. Cold Spring Harb Perspect Biol, 2016, 9(2): a022129.
36.	Lee J, Moon HJ, Lee JM, et al. Smad3 regulates Rho signaling via NET1 in the transforming growth factor-β-induced epithelial-mesenchymal transition of human retinal pigment epithelial cells. J Biol Chem, 2010, 285(34): 26618-26627.
37.	Ozdamar B, Bose R, Barrios-Rodiles M, et al. Regulation of the polarity protein Par6 by TGFβ receptors controls epithelial cell plasticity. Science, 2005, 307(5715): 1603-1609.

1. Bousquet J, Jeffery PK, Busse WW, et al. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med, 2000, 161(5): 1720-1745.
2. Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease. Cell, 2009, 139(5): 871-890.
3. Lee JM, Dedhar S, Kalluri R, et al. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol, 2006, 172(7): 973-981.
4. Sohal SS, Walters EH. Role of epithelial mesenchymal transition (EMT) in chronic obstructive pulmonary disease (COPD). Respir Res, 2013, 14: 120.
5. Nowrin K, Sohal SS, Peterson G, et al. Epithelial-mesenchymal transition as a fundamental underlying pathogenic process in COPD airways: fibrosis, remodeling and cancer. Expert Rev Respir Med, 2014, 8(5): 547-559.
6. Heijink IH, Postma DS, Noordhoek JA, et al. House dust mite-promoted epithelial-to-mesenchymal transition in human bronchial epithelium. Am J Respir Cell Mol Biol, 2010, 42(1): 69-79.
7. Haynes J, Srivastava J, Madson N, et al. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression. Mol Biol Cell, 2011, 22(24): 4750-4764.
8. Fehon RG, McClatchey AI, Bretscher A. Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol, 2010, 11(4): 276-287.
9. Moleirinho S, Tilston-Lunel A, Angus L, et al. The expanding family of FERM proteins. Biochem J, 2013, 452(2): 183-193.
10. Valderrama F, Thevapala S, Ridley AJ. Radixin regulates cell migration and cell-cell adhesion through Rac1. J Cell Sci, 2012, 125(Pt 14): 3310-3319.
11. Chiappetta C, Leopizzi M, Censi F, et al. Correlation of the Rac1/RhoA pathway with ezrin expression in osteosarcoma. Appl Immunohistochem Mol Morphol, 2014, 22(3): 162-170.
12. Arumugam P, Partelli S, Coleman SJ, et al. Ezrin expression is an independent prognostic factor in gastro-intestinal cancers. J Gastrointest Surg, 2013, 17(12): 2082-2091.
13. Kong J, Li Y, Liu S, et al. High expression of ezrin predicts poor prognosis in uterine cervical cancer. BMC Cancer, 2013, 13: 520.
14. Leiphrakpam PD, Rajput A, Mathiesen M, et al. Ezrin expression and cell survival regulation in colorectal cancer. Cell Signal, 2014, 26(5): 868-879.
15. Ren L, Hong SH, Cassavaugh J, et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene, 2009, 28(6): 792-802.
16. Bretscher A. Purification of an 80, 000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J Cell Biol, 1983, 97(2): 425-432.
17. Xie JJ, Zhang FR, Tao LH, et al. Expression of ezrin in human embryonic, fetal, and normal adult tissues. J Histochem Cytochem, 2011, 59(11): 1001-1008.
18. Bonilha VL. Focus on molecules: ezrin. Exp Eye Res, 2007, 84(4): 613-614.
19. Berryman M, Franck Z, Bretscher A. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci, 1993, 105(Pt 4): 1025-1043.
20. Mangeat P, Roy C, Martin M. ERM proteins in cell adhesion and membrane dynamics. Trends Cell Biol, 1999, 9(5): 187-192.
21. Di Cristofano C, Leopizzi M, Miraglia A, et al. Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell. Mod Pathol, 2010, 23(7): 1012-1020.
22. Sarrió D, Rodríguez-Pinilla SM, Dotor A, et al. Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast Cancer Res Treat, 2006, 98(1): 71-79.
23. Tokunou M, Niki T, Saitoh Y, et al. Altered expression of the ERM proteins in lung adenocarcinoma. Lab Invest, 2000, 80(11): 1643-1650.
24. Chaw SY, Majeed AA, Dalley AJ, et al. Epithelial to mesenchymal transition (EMT) biomarkers--E-cadherin, beta-catenin, APC and Vimentin--in oral squamous cell carcinogenesis and transformation. Oral Oncol, 2012, 48(10): 997-1006.
25. Araki K, Ebata T, Guo AK, et al. p53 regulates cytoskeleton remodeling to suppress tumor progression. Cell Mol Life Sci, 2015, 72(21): 4077-4094.
26. McVicker CG, Leung SY, Kanabar V, et al. Repeated allergen inhalation induces cytoskeletal remodeling in smooth muscle from rat bronchioles. Am J Respir Cell Mol Biol, 2007, 36(6): 721-727.
27. Sahai E, Marshall CJ. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol, 2003, 5(8): 711-719.
28. Aubert JD, Dalal BI, Bai TR, et al. Transforming growth factor β1 gene expression in human airways. Thorax, 1994, 49(3): 225-232.
29. Zavadil J, Böttinger EP. TGF-β and epithelial-to-mesenchymal transitions. Oncogene, 2005, 24(37): 5764-5774.
30. Vega G, Alarcón S, San Martín R. The cellular and signalling alterations conducted by TGF-β contributing to renal fibrosis. Cytokine, 2016, 88: 115-125.
31. O'Connor JW, Riley PN, Nalluri SM, et al. Matrix rigidity mediates TGFβ1-induced epithelial-myofibroblast transition by controlling cytoskeletal organization and MRTF-A localization. J Cell Physiol, 2015, 230(8): 1829-1839.
32. Chen MJ, Gao XJ, Xu LN, et al. Ezrin is required for epithelial-mesenchymal transition induced by TGF-β 1 in A549 cells. Int J Oncol, 2014, 45(4): 1515-1522.
33. Mani SA, Guo W, Liao MJ, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell, 2008, 133(4): 704-715.
34. Phanish MK, Wahab NA, Colville-Nash P, et al. The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells. Biochem J, 2006, 393(Pt 2): 601-607.
35. Zhang YE. Non-Smad signaling pathways of the TGF-β family. Cold Spring Harb Perspect Biol, 2016, 9(2): a022129.
36. Lee J, Moon HJ, Lee JM, et al. Smad3 regulates Rho signaling via NET1 in the transforming growth factor-β-induced epithelial-mesenchymal transition of human retinal pigment epithelial cells. J Biol Chem, 2010, 285(34): 26618-26627.
37. Ozdamar B, Bose R, Barrios-Rodiles M, et al. Regulation of the polarity protein Par6 by TGFβ receptors controls epithelial cell plasticity. Science, 2005, 307(5715): 1603-1609.

Chinese Journal of Respiratory and Critical Care Medicine

The effects of ezrin on transforming growth factor beta mediated epithelial mesenchymal transition

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