Skip to main content

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

Abstract

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

Abbreviations

α-SMA:

α-smooth muscle actin

CK19:

cytokeratin19

E-cad:

E-cadherin

ECM:

extracellular matrix

EGFR:

epidermal growth factor receptor

EMT:

epithelial-mesenchymal transition

FN:

fibronectin

IPF:

Idiopathic pulmonary fibrosis

TGF-β1:

transforming growth factor-β1

References

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Google Scholar 

  3. Selman, M. and Pardo, A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respir Res. 3 (2002) 3.

    PubMed  Article  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  5. Gauldie, J., Kolb, M. and Sime, P.J. A new direction in the pathogenesis of idiopathic pulmonary fibrosis? Respir Res. 3 (2002) 1.

    PubMed  Article  Google Scholar 

  6. Kalluri, R. and Neilson, E.G. Epithelial-mesenchymal transition and its implications for fibrosis. J. Clin. Invest. 112 (2003) 1776–1784.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  10. Desmouliere, A. Factors influencing myofibroblast differentiation during wound healing and fibrosis. Cell Biol. Int. 19 (1995) 471–476.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  12. Massague, J. and Wotton, D. Transcriptional control by the TGF-b/Smad signaling system. EMBO J. 19 (2000) 1745–1754.

    PubMed  Article  CAS  Google Scholar 

  13. Derynck, R. and Zhang, Y.E. Smad-dependent and Smad-independent pathways in TGF-b family signaling. Nature 425 (2003) 577–584.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  16. Zavadil, J. and Bottinger, E.P. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24 (2005) 5764–5774.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  18. Stoker, M. and Perryman, M. An epithelial scatter factor released by embryo fibroblasts. J. Cell Sci. 77 (1985) 209–23.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  20. Kalluri, R. and Neilson, E.G. Epithelial-mesenchymal transition and its implications for fibrosis. J. Clin. Invest. 112 (2003) 1776–1784.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zu-De Xu.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xu, GP., Li, QQ., Cao, XX. et al. The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells. Cell Mol Biol Lett 12, 457–472 (2007). https://doi.org/10.2478/s11658-007-0018-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-007-0018-x

Keywords

  • Epithelial-mesenchymal transition
  • Gene transfer
  • Smad7
  • Transforming growth factor-β1