Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression.
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-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta 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-beta. 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-beta signal pathway that did not involve Smad4.
Imamura
Takaaki
T
Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Kanai
Fumihiko
F
Kawakami
Takayuki
T
Amarsanaa
Jazag
J
Ijichi
Hideaki
H
Hoshida
Yujin
Y
Tanaka
Yasuo
Y
Ikenoue
Tsuneo
T
Tateishi
Keisuke
K
Kawabe
Takao
T
Arakawa
Yoshihiro
Y
Miyagishi
Makoto
M
Taira
Kazunari
K
Yokosuka
Osamu
O
Omata
Masao
M
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D020868:Gene Silencing; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D015398:Signal Transduction; D051901:Smad4 Protein; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011506:Proteins; D020543:Proteome; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one