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Tumor Biology

, Volume 36, Issue 5, pp 3643–3651 | Cite as

MAPK inhibitors differently modulate TGF-β/Smad signaling in HepG2 cells

Research Article

Abstract

The aim of this study was to investigate the mitogen-activated protein kinase (MAPK) pathway, which crosstalk with TGF-β/Smad signaling via linker phosphorylation of Smad2/3 to promote hepatocarcinogenesis. After DEN-induced hepatocellular carcinoma (HCC) in rats showed increased phosphorylation of JNK1/2, p38, and ERK1/2, we next antagonized TGF-β1-induced phosphorylation of JNK1/2, p38, ERK1/2, Smad2/3 signaling in HepG2 cells using SP600125, SB203580, and PD98059, respectively. Cell proliferation and invasion were assessed by MTT assay and transwell invasion chambers, respectively. Smad2/3, Smad4, and Smad7 expressions and PAI-1 messenger RNA (mRNA) transcription were measured by using immuno-precipitation/immuno-blotting and real-time RT-PCR, respectively. All the MAPK-specific inhibitors suppressed cell invasion, while all but PD98059 suppressed cell proliferation. Both SP600125 and SB203580 blocked pSmad2C/L and oncogenic pSmad3L. PD98059 blocked pSmad2L but had no effect on elevated pSmad2C and oncogenic pSmad3L. All but PD98059 blocked Smad2/3/4 complex formation and restored Smad7 expression, while all the three MAPK-Specific inhibitors repressed PAI-1 mRNA transcription. Both SP600125 and SB203580 inhibited HepG2 cells’ proliferation and invasion by blocking oncogenic pSmad3L and Smad2/3/4 complex formation. PD98059 repressed PAI-1 mRNA by an unknown mechanism.

Keywords

Hepatocellular carcinoma MAPK-specific inhibitors Smad TGF-β1 

Notes

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (no. 81073098, no. 81374012). We also thank Prof. K Matsuzaki, Department of Gastroenterology and Hepatology, Kansai Medical University, Osaka, Japan, for providing us the following Abs: anti-pSmad2L and anti-pSmad3L.

Conflicts of interest

None

References

  1. 1.
    Derynck R, Akhurst RJ, Balmain A. TGF-β signaling in tumor suppression and cancer progression. Nat Genet. 2001;29:117–29.CrossRefPubMedGoogle Scholar
  2. 2.
    Wakefield LM, Roberts AB. TGF-β signaling: positive and negative effects on tumorigenesis. Curr Opinion Genet Dev. 2002;12:22–9.CrossRefGoogle Scholar
  3. 3.
    Derynck R, Akhurst RJ. Differentiation plasticity regulated by TGF-β family proteins in development and disease. Nat Cell Biol. 2007;9:1000–4.CrossRefPubMedGoogle Scholar
  4. 4.
    Engel ME, McDonnell MA, Law BK, Moses HL. Interdependent SMAD and JNK signaling in transforming growth factor-β-mediated transcription. J Biol Chem. 1999;274:37413–20.CrossRefPubMedGoogle Scholar
  5. 5.
    Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J-i, Shibuya H, et al. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-β-induced gene expression. J Biol Chem. 1999;274:27161–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Javelaud D, Mauviel A. Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-β: implications for carcinogenesis. Oncogene. 2005;24:5742–50.CrossRefPubMedGoogle Scholar
  7. 7.
    Yoshida K, Murata M, Yamaguchi T, Matsuzaki K. TGF-β/Smad signaling during hepatic fibro-carcinogenesis (review). Int J Oncol. 2014;45:1363–71.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Velden JL, Alcorn JF, Guala AS, Badura EC, Janssen-Heininger YM. c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3. Am J Respir Cell Mol Biol. 2011;44:571–81.CrossRefPubMedGoogle Scholar
  9. 9.
    Hu X, Rui W, Wu C, He S, Jiang J, Zhang X, et al. Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-β/Smad signaling. J Gastroenterol Hepatol. 2014;29:1284–91.CrossRefPubMedGoogle Scholar
  10. 10.
    Liu X, Yang Y, Zhang X, Xu S, He S, Huang W, et al. Compound Astragalus and Salvia miltiorrhiza extract inhibits cell invasion by modulating transforming growth factor-β/Smad in HepG2 cell. J Gastroenterol Hepatol. 2010;25:420–6.CrossRefPubMedGoogle Scholar
  11. 11.
    Rui W, Xie L, Liu X, He S, Wu C, Zhang X, et al. Compound Astragalus and Salvia miltiorrhiza extract suppresses hepatocellular carcinoma progression by inhibiting fibrosis and PAI-1 mRNA transcription. J Ethnopharmacol. 2014;151:198–209.CrossRefPubMedGoogle Scholar
  12. 12.
    He S, Liu X, Yang Y, Huang W, Xu S, Yang S, et al. Mechanisms of transforming growth factor β1/Smad signalling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts. Br J Dermatol. 2010;162:538–46.CrossRefPubMedGoogle Scholar
  13. 13.
    Mori S, Matsuzaki K, Yoshida K, Furukawa F, Tahashi Y, Yamagata H, et al. TGF-β and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions. Oncogene. 2004;23:7416–29.CrossRefPubMedGoogle Scholar
  14. 14.
    Giehl K, Imamichi Y, Menke A. Smad4-independent TGF-β signaling in tumor cell migration. Cells Tissues Organs. 2007;185:123–30.CrossRefPubMedGoogle Scholar
  15. 15.
    Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y, et al. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology. 2003;38:879–89.CrossRefPubMedGoogle Scholar
  16. 16.
    Kamaraju AK, Roberts AB. Role of Rho/ROCK and p38 map kinase pathways in transforming growth factor-β-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo. J Biol Chem. 2005;280:1024–36.CrossRefPubMedGoogle Scholar
  17. 17.
    Kretzschmar M, Doody J, Timokhina I, Massagué J. A mechanism of repression of TGFβ/Smad signaling by oncogenic Ras. Genes Dev. 1999;13:804–16.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Nagata H, Hatano E, Tada M, Murata M, Kitamura K, Asechi H, et al. Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor-suppression in rat hepatocellular carcinoma. Hepatology. 2009;49:1944–53.CrossRefPubMedGoogle Scholar
  19. 19.
    Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development. Curr Opin Cell Biol. 1998;10:205–19.CrossRefPubMedGoogle Scholar
  20. 20.
    Wang G, Long J, Matsuura I, He D, Liu F. The Smad3 linker region contains a transcriptional activation domain. Biochem J. 2005;386:29–34.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Matsuzaki K. Smad phosphoisoform signals in acute and chronic liver injury: Similarities and differences between epithelial and mesenchymal cells. Cell Tissue Res. 2012;347:225–43.CrossRefPubMedGoogle Scholar
  22. 22.
    Matsuzaki K, Kitano C, Murata M, Sekimoto G, Yoshida K, Uemura Y, et al. Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-β signal in later stages of human colorectal cancer. Cancer Res. 2009;69:5321–30.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhang H, Ozaki I, Mizuta T, Yoshimura T, Matsuhashi S, Eguchi Y, et al. Transforming growth factor-β1-induced apoptosis is blocked by β1-integrin-mediated mitogen-activated protein kinase activation in human hepatoma cells. Cancer Sci. 2004;95:878–86.CrossRefPubMedGoogle Scholar
  24. 24.
    Park HJ, Kim BC, Kim SJ, Choi KS. Role of MAP kinases and their cross-talk in TGF-β1–induced apoptosis in FaO rat hepatoma cell line. Hepatology. 2002;35:1360–71.CrossRefPubMedGoogle Scholar
  25. 25.
    Yang Y, Zhou F, Fang Z, Wang L, Li Z, Sun L, et al. Post-transcriptional and post-translational regulation of PTEN by transforming growth factor-β1. J Cell Biochem. 2009;106:1102–12.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Department of Pharmacology and Institute of Natural MedicineAnhui Medical UniversityHefeiChina
  2. 2.Department of AnesthesiologyThe Second Hospital of Anhui Medical UniversityHefeiChina

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