The effect of TGF-beta1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

• Autorzy: Xu G P , Li Q.Q. , Cao X.X. , Chen Q. , Zhao Z.H. , Diao Z.Q. , Xu Z.D.
• Języki publikacji: EN

Abstrakty

EN

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process.

Słowa kluczowe

EN

gene transfer; epithelial cell; Smad7 gene; epithelial-mesenchymal transition; extracellular matrix; E-cadherin; fibronectin; epidermal growth factor receptor; transforming growth factor-beta1; idiopathic pulmonary fibrosis

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2007
• Tom: 12
• Numer: 3
• Opis fizyczny: p.457-472,fig.,ref.

Twórcy

autor

Xu G P

• Fudan University, Shanghai 200032, China

autor

Li Q.Q.

autor

Cao X.X.

autor

Chen Q.

autor

Zhao Z.H.

autor

Diao Z.Q.

autor

Xu Z.D.

Bibliografia

• 1. Selman, M., King, T.E. and Pardo, A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134 (2001) 136-151.
• 2. King, T.E., Schwarz, M.I., Brown, K., Tooze, J.A., Colby, T.V., Waldron, J.A., Flint, A., Thurlbeck, W. and Cherniack, R.M. Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am. J. Respir. Crit. Care Med. 164 (2001) 1025-1032.
• 3. Selman, M. and Pardo, A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respir Res. 3 (2002) 3.
• 4. Yao, H.W., Xie, Q.M., Chen, J.Q., Deng, Y.M. and Tang, H.F. TGF-beta 1 induces alveolar epithelial to mesenchymal cell transition in vitro. Life Sci. 76 (2004) 29-37.
• 5. Gauldie, J., Kolb, M. and Sime, P.J. A new direction in the pathogenesis of idiopathic pulmonary fibrosis? Respir Res. 3 (2002) 1.
• 6. Kalluri, R. and Neilson, E.G. Epithelial-mesenchymal transition and its implications for fibrosis. J. Clin. Invest. 112 (2003) 1776-1784.
• 7. Liu, Y. Epithelial to mesenchymal transition in renal fibrogenesis: athologic significance, molecular mechanism, and therapeutic intervention. J. Am. Soc. Nephrol. 15 (2004) 1-12.
• 8. Desmouliere, A., Darby, I.A. and Gabbiani, G. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab. Invest. 83 (2003) 1689-1707.
• 9. Moustakas, A., Pardali, K. and Gaal, A. Mechanisms of TGF-b signaling in regulation of cell growth and differentiation. Immunol. Lett. 82 (2002) 85-91.
• 10. Desmouliere, A. Factors influencing myofibroblast differentiation during wound healing and fibrosis. Cell Biol. Int. 19 (1995) 471-476.
• 11. ten Dijke, P., Goumans, M.J., Itoh, F. and Itoh, S. Regulation of cell proliferation by Smad proteins. J. Cell Physiol. 191 (2002) 1-16.
• 12. Massague, J. and Wotton, D. Transcriptional control by the TGF-b/Smad signaling system. EMBO J. 19 (2000) 1745-1754.
• 13. Derynck, R. and Zhang, Y.E. Smad-dependent and Smad-independent pathways in TGF-b family signaling. Nature 425 (2003) 577-584.
• 14. Lan, H.Y., Mu,W., Tomita, N., Huang, X.R., Li, J.H. and Zhu, H.J. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using Ultrasound-microbubble system in rat UUO model. J. Am. Soc. Nephrol. 14 (2003) 1535-1548.
• 15. Dooley, S., Hamzavi, J., Breitkopf, K., Wiercinska, E., Said, H.M., Lorenzen, J., ten Dijke, P. and Gressner, A.M. Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 125 (2003) 178-191.
• 16. Zavadil, J. and Bottinger, E.P. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24 (2005) 5764-5774.
• 17. Greenburg, G. and Hay, E.D. Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. J. Cell Biol. 95 (1982) 333-339.
• 18. Stoker, M. and Perryman, M. An epithelial scatter factor released by embryo fibroblasts. J. Cell Sci. 77 (1985) 209-23.
• 19. Miettinen, P.J., Ebner, R., Lopez, A.R. and Derynck, R. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J. Cell Biol. 127 (1994) 2021-2036.
• 20. Kalluri, R. and Neilson, E.G. Epithelial-mesenchymal transition and its implications for fibrosis. J. Clin. Invest. 112 (2003) 1776-1784.
• 21. Saika, S., Kono-Saika, S., Tanaka, T., Yamanaka, O., Ohnishi, Y., Sato, M., Muragaki, Y., Ooshima, A., Yoo, J., Flanders, K.C. and Roberts, A.B. Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice. Lab. Invest. 84 (2004) 1245-1258.
• 22. Valcourt, U., Kowanetz, M., Niimi, H., Valcourt U., Heldin, C.H. and Moustakas, A. TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol. Biol. Cell. 16 (2005) 1987-2002.
• 23. Lan, H.Y., Mu, W., Tomita, N., Huang, X.R., Li, J.H., Zhu, H.J., Morishita, R. and Johnson, R.J. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J. Am. Soc. Nephrol. 14 (2003) 1535-1548.