Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells.
Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling.
Key Laboratory of Swine Breeding and Genetics, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China.
Rong
R
Zheng
Qi
Q
Xiong
Bo
B
Zuo
SiWen
S
Jiang
FengE
F
Li
MingGang
M
Lei
ChangYan
C
Deng
YuanZhu
Y
Xiong
D000818:Animals; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D034622:RNA Interference; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051900:Smad3 Protein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology
D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D053773:Transforming Growth Factor beta1