Proteomic analysis of the TGF-β signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression

Takaaki Imamura, Fumihiko Kanai, Takayuki Kawakami, Jazag Amarsanaa, Hideaki Ijichi, Yujin Hoshida, Yasuo Tanaka, Tsuneo Ikenoue, Keisuke Tateishi, Takao Kawabe, Yoshihiro Arakawa, Makoto Miyagishi, Kazunari Taira, Osamu Yokosuka, Masao Omata

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-β (TGF-β) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-β-induced growth inhibition, there seems to be a Smad4-independent TGF-β signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-β-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-β stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-β. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-β signal pathway that did not involve Smad4.

Original languageEnglish (US)
Pages (from-to)289-296
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume318
Issue number1
DOIs
StatePublished - May 21 2004
Externally publishedYes

Keywords

  • 2-DE
  • Proteomics
  • Smad4
  • Stable RNAi
  • TGF-β

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Imamura, T., Kanai, F., Kawakami, T., Amarsanaa, J., Ijichi, H., Hoshida, Y., Tanaka, Y., Ikenoue, T., Tateishi, K., Kawabe, T., Arakawa, Y., Miyagishi, M., Taira, K., Yokosuka, O., & Omata, M. (2004). Proteomic analysis of the TGF-β signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression. Biochemical and Biophysical Research Communications, 318(1), 289-296. https://doi.org/10.1016/j.bbrc.2004.04.029