Advertisement

Molecular and Cellular Biochemistry

, Volume 340, Issue 1–2, pp 21–29 | Cite as

Focal adhesion kinase mediates TGF-β1-induced renal tubular epithelial-to-mesenchymal transition in vitro

  • Bingqing Deng
  • Xiao Yang
  • Jianshe Liu
  • Fangfang He
  • Zhonghua Zhu
  • Chun Zhang
Article

Abstract

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which participates in many important cellular processes such as cell adhesion and migration. However, the role of FAK in renal tubular epithelial-to-mesenchymal transition (EMT) is still unknown. FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-β1). The expression of FAK, α-smooth muscle actin (α-SMA),E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9),tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV were detected by RT-PCR, Western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay. The results suggest that the expression of FAK was up-regulated in HK-2 cells when incubated with TGF-β1(10 μg/l), which was accompanied by reduced expression of E-cadherin and increased expression of α-SMA. All these changes were restored by FAK siRNA. Akt phosphorylation was induced by the treatment with TGF-β1, which was blocked by FAK siRNA. TGF-β1-induced down-regulation of E-cadherin was recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-β1 induced an increase in MMP-9 expression, as well as decreased expression of TIMP-1 and collagen IV, which were all restored by the FAK siRNA transfection. In addition, FAK siRNA significantly reduced TGF-β1-induced cells migration. In conclusion, FAK may play a crucial role in mediating TGF-β1-induced EMT through the activation of Akt pathway.

Keywords

Focal adhesion kinase Transforming growth factor-beta 1 Epithelial-to-mesenchymal transition Renal tubule 

Notes

Acknowledgments

This study was supported by grants from National Natural Science Foundation of China (No.30871174).

References

  1. 1.
    Hay ED, Zuk A (1995) Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced. Am J Kidney Dis 26:678–690CrossRefPubMedGoogle Scholar
  2. 2.
    Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172:973–981CrossRefPubMedGoogle Scholar
  3. 3.
    Nieto MA (2002) The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 3:155–166CrossRefPubMedGoogle Scholar
  4. 4.
    Zhang C, Meng X, Zhu Z, Liu J, Deng A (2004) Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biol Int 28:863–873CrossRefPubMedGoogle Scholar
  5. 5.
    Zeng ZZ, Jia Y, Hahn NJ, Markwart SM, Rockwood KF, Livant DL (2006) Role of focal adhesion kinase and phosphatidylinositol 3′-kinase in integrin fibronectin receptor-mediated, matrix metalloproteinase-1-dependent invasion by metastatic prostate cancer cells. Cancer Res 66:8091–8099CrossRefPubMedGoogle Scholar
  6. 6.
    McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC (2005) The role of focal-adhesion kinase in cancer––a new therapeutic opportunity. Nat Rev Cancer 5:505–515CrossRefPubMedGoogle Scholar
  7. 7.
    Planas-Silva MD, Bruggeman RD, Grenko RT, Stanley Smith J (2006) Role of c-src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer. Biochem Biophys Res Commun 341:73–81CrossRefPubMedGoogle Scholar
  8. 8.
    Han EK, McGonigal T (2007) Role of focal adhesion kinase in human cancer: a potential target for drug discovery. Anticancer Agents Med Chem 7:681–684CrossRefPubMedGoogle Scholar
  9. 9.
    Liu G, Meng X, Jin Y, Bai J, Zhao Y, Cui X, Chen F, Fu S (2008) Inhibitory role of focal adhesion kinase on anoikis in the lung cancer cell a549. Cell Biol Int 32:663–670CrossRefPubMedGoogle Scholar
  10. 10.
    Yano H, Mazaki Y, Kurokawa K, Hanks SK, Matsuda M, Sabe H (2004) Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. J Cell Biol 166:283–295CrossRefPubMedGoogle Scholar
  11. 11.
    Zavadil J, Bottinger EP (2005) Tgf-beta and epithelial-to-mesenchymal transitions. Oncogene 24:5764–5774CrossRefPubMedGoogle Scholar
  12. 12.
    Nakamura K, Yano H, Schaefer E, Sabe H (2001) Different modes and qualities of tyrosine phosphorylation of fak and pyk2 during epithelial-mesenchymal transdifferentiation and cell migration: analysis of specific phosphorylation events using site-directed antibodies. Oncogene 20:2626–2635CrossRefPubMedGoogle Scholar
  13. 13.
    Repesh LA (1989) A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9:192–208PubMedGoogle Scholar
  14. 14.
    Yang J, Liu Y (2001) Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 159:1465–1475PubMedGoogle Scholar
  15. 15.
    Fan JM, Ng YY, Hill PA, Nikolic-Paterson DJ, Mu W, Atkins RC, Lan HY (1999) Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 56:1455–1467CrossRefPubMedGoogle Scholar
  16. 16.
    Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT (1992) Pp125fak a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA 89:5192–5196CrossRefPubMedGoogle Scholar
  17. 17.
    Gilmore AP, Romer LH (1996) Inhibition of focal adhesion kinase (fak) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell 7:1209–1224PubMedGoogle Scholar
  18. 18.
    Salazar EP, Rozengurt E (1999) Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with src in intact swiss 3t3 cells. J Biol Chem 274:28371–28378CrossRefPubMedGoogle Scholar
  19. 19.
    Yu CF, Basson MD (2000) Matrix-specific fak and mapk reorganization during caco-2 cell motility. Microsc Res Tech 51:191–203CrossRefPubMedGoogle Scholar
  20. 20.
    Watermann DO, Gabriel B, Jager M, Orlowska-Volk M, Hasenburg A, zur Hausen A, Gitsch G, Stickeler E (2005) Specific induction of pp125 focal adhesion kinase in human breast cancer. Br J Cancer 93:694–698CrossRefPubMedGoogle Scholar
  21. 21.
    Gabarra-Niecko V, Schaller MD, Dunty JM (2003) Fak regulates biological processes important for thepathogenesis of cancer. Cancer Metastasis Rev 22:359–374CrossRefPubMedGoogle Scholar
  22. 22.
    Walsh MF, Ampasala DR, Hatfield J, Vander Heide R, Suer S, Rishi AK, Basson MD (2008) Transforming growth factor-beta stimulates intestinal epithelial focal adhesion kinase synthesis via smad-and p38-dependent mechanisms. Am J Pathol 173:385–399CrossRefPubMedGoogle Scholar
  23. 23.
    Cicchini C, Laudadio I, Citarella F, Corazzari M, Steindler C, Conigliaro A, Fantoni A, Amicone L, Tripodi M (2008) TGFbeta-induced emt requires focal adhesion kinase (fak) signaling. Exp Cell Res 314:143–152CrossRefPubMedGoogle Scholar
  24. 24.
    Sundberg LJ, Galante LM, Bill HM, Mack CP, Taylor JM (2003) An endogenous inhibitor of focal adhesion kinase blocks rac1/jnk but not ras/erk-dependent signaling in vascular smooth muscle cells. J Biol Chem 278:29783–29791CrossRefPubMedGoogle Scholar
  25. 25.
    Maa MC, Lee JC, Chen YJ, Lee YC, Wang ST, Huang CC, Chow NH, Leu TH (2007) Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase. J Biol Chem 282:19399–19409CrossRefPubMedGoogle Scholar
  26. 26.
    Beierle EA, Trujillo A, Nagaram A, Kurenova EV, Finch R, Ma X, Vella J, Cance WG, Golubovskaya VM (2007) N-myc regulates focal adhesion kinase expression in human neuroblastoma. J Biol Chem 282:12503–12516CrossRefPubMedGoogle Scholar
  27. 27.
    Franchini KG, Torsoni AS, Soares PH, Saad MJ (2000) Early activation of the multicomponent signaling complex associated with focal adhesion kinase induced by pressure overload in the rat heart. Circ Res 87:558–565PubMedGoogle Scholar
  28. 28.
    Kim RD, Darling CE, Roth TP, Ricciardi R, Chari RS (2001) Activator protein 1 activation following hypoosmotic stress in hepg2 cells is actin cytoskeleton dependent. J Surg Res 100:176–182CrossRefPubMedGoogle Scholar
  29. 29.
    Koshida R, Rocic P, Saito S, Kiyooka T, Zhang C, Chilian WM (2005) Role of focal adhesion kinase in flow-induced dilation of coronary arterioles. Arterioscler Thromb Vasc Biol 25:2548–2553CrossRefPubMedGoogle Scholar
  30. 30.
    Larue L, Bellacosa A (2005) Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/akt pathways. Oncogene 24:7443–7454CrossRefPubMedGoogle Scholar
  31. 31.
    Grille SJ, Bellacosa A, Upson J, Klein-Szanto AJ, van Roy F, Lee-Kwon W, Donowitz M, Tsichlis PN, Larue L (2003) The protein kinase akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res 63:2172–2178PubMedGoogle Scholar
  32. 32.
    Zeisberg M, Maeshima Y, Mosterman B, Kalluri R (2002) Renal fibrosis. Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells. Am J Pathol 160:2001–2008PubMedGoogle Scholar
  33. 33.
    Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17:463–516CrossRefPubMedGoogle Scholar
  34. 34.
    Jiang MC, Liao CF, Lee PH (2001) Aspirin inhibits matrix metalloproteinase-2 activity, increases e-cadherin production, and inhibits in vitro invasion of tumor cells. Biochem Biophys Res Commun 282:671–677CrossRefPubMedGoogle Scholar
  35. 35.
    Chambers AF, Matrisian LM (1997) Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 89:1260–1270CrossRefPubMedGoogle Scholar
  36. 36.
    Wang M, Liu YE, Greene J, Sheng S, Fuchs A, Rosen EM, Shi YE (1997) Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4. Oncogene 14:2767–2774CrossRefPubMedGoogle Scholar
  37. 37.
    Murphy G, Gavrilovic J (1999) Proteolysis and cell migration: creating a path? Curr Opin Cell Biol 11:614–621CrossRefPubMedGoogle Scholar
  38. 38.
    Yang J, Shultz RW, Mars WM, Wegner RE, Li Y, Dai C, Nejak K, Liu Y (2002) Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy. J Clin Invest 110:1525–1538PubMedGoogle Scholar
  39. 39.
    Fujii T, Koshikawa K, Nomoto S, Okochi O, Kaneko T, Inoue S, Yatabe Y, Takeda S, Nakao A (2004) Focal adhesion kinase is overexpressed in hepatocellular carcinoma and can be served as an independent prognostic factor. J Hepatol 41:104–111CrossRefPubMedGoogle Scholar
  40. 40.
    Caceres M, Guerrero J, Martinez J (2005) Overexpression of rhoa-gtp induces activation of the epidermal growth factor receptor, dephosphorylation of focal adhesion kinase and increased motility in breast cancer cells. Exp Cell Res 309:229–238CrossRefPubMedGoogle Scholar
  41. 41.
    Cary LA, Chang JF, Guan JL (1996) Stimulation of cell migration by overexpression of focal adhesion kinase and its association with src and fyn. J Cell Sci 109:1787–1794PubMedGoogle Scholar
  42. 42.
    Reiske HR, Kao SC, Cary LA, Guan JL, Lai JF, Chen HC (1999) Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. J Biol Chem 274:12361–12366CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  1. 1.Department of Nephrology, Union Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina

Personalised recommendations