Identification of the gene transcription mediated by TGF-β-Smad2/3-Smad4 signaling

TGF-β signaling is known to be very much dependent on the formation of Smad2/3-Smad4 transcription regulatory complexes. However, the signaling functions of Smad2/3-Smad4 in TGF-β-induced responses are obscure as TGF-β also initiates a number of other signaling pathways. In this study, we systematically assessed the contribution of TGF-β-Smad2/3-Smad4 signaling to target gene transcription. Individual Smads were selectively knocked down in Hep3B cells by stable RNA interference (RNAi). We identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2, Smad3 or Smad4 by the combination of RNAi and microarray assay. The major finding from our microarray analysis was that of the 2039 target genes seen to be regulated via TGF-β induction, 190 were differentially transcriptionally controlled by Smad2-Smad4 and Smad3-Smad4 signaling and the latter control mechanism appeared to be functionally more important. We also found evidence of competition between Smad2 and Smad3 for their activation when controlling the transcription of target genes. Keywords: cell type comparison Large-scale microarray analysis was employed for comparison between the responses of wild-type Hep3B cell line and each knockdown cell line, with and without TGF-β induction, to identify target genes and any false positive genes.