Abstract
Cited2 is a transcription factor without typical DNA binding domains. Cited2 interacts with cAMP-responsive element-binding protein-binding protein (CBP)/p300, TFAP2, Lhx2, and nuclear receptors, such as peroxisome proliferator-activated receptor and estrogen receptor to function as a transcriptional modulator. Overexpression of Cited2 in Rat1 cells leads to tumor formation in nude mice, suggesting that Cited2 is a transforming gene. Through microarray analysis, Cited2 was found to be down-regulated by transforming growth factor beta1 (TGF-beta) in various cell lines. In this study, we confirmed that both mRNA and protein levels of Cited2 are down-regulated in MDA-MB-231 breast cancer cells. Overexpression of Smad7 or knockdown of Smad4 in MDA-MB-231 cells showed that the Smad pathway is involved in the down-regulation of Cited2. Based on nuclear run-on analysis and Cited2 promoter/reporter assay, Cited2 transcription was not affected by TGF-beta, supporting that down-regulation of Cited2 by TGF-beta is most likely through post-transcriptional regulation. By using transcriptional inhibitors, we demonstrated that the turnover of Cited2 transcripts appears to be accelerated during TGF-beta stimulation. Pharmacologic inhibition of translation with cycloheximide attenuated Cited2 down-regulation by TGF-beta. We examined the expression of recombinant Cited2 gene introduced into MDA-MB-231 cells by stable transfection, and we found that mRNA containing the Cited2 protein-coding region controlled by a heterologous promoter indeed responds to TGF-beta-mediated down-regulation. Study from Cited2 deletion mutants showed that the C-terminal conserved region of Cited2 coding sequence is essential for the down-regulation. This is the first demonstration that TGF-beta-mediated down-regulation of Cited2 is post-transcriptional, through the Smad pathway, and requires the presence of its coding sequence.
Highlights
Cited2 (cAMP-responsive element-binding protein-binding protein (CBP)2/p300-interacting transactivators with glutamic acid and aspartic acid-rich tail) is one of the founding members of transcriptional activators, previously named melanocyte-specific gene-related gene (MRG)1/p35srj [1,2,3,4]
By stable expression of the Cited2 coding sequence under the control of a heterologous promoter, we found that the expression of endogenous and exogenous Cited2 mRNA is downregulated by TGF-, suggesting that the Cited2 coding sequence is sufficient for proper regulation
TGF- Down-regulates Cited2 in MDA-MB-231 Cells—Chen et al [26] showed previously by microarray analysis that Cited2 is one of the genes down-regulated by TGF- in both MCF-10A and MDA-MB231 cells
Summary
Cited2 (cAMP-responsive element-binding protein-binding protein (CBP)2/p300-interacting transactivators with glutamic acid and aspartic acid-rich tail) is one of the founding members of transcriptional activators, previously named melanocyte-specific gene-related gene (MRG)1/p35srj [1,2,3,4]. To evaluate whether the Smad pathway is involved in the down-regulation of Cited2 by TGF-, an expression plasmid for Smad7, an inhibitory Smad, was introduced into MDA-MB-231 cells through retrovirus-mediated gene transfer (Fig. 2A). Cotransfection with both si-Smad2 and si-Smad3 effectively decreased TGF--mediated downregulation of Cited2 mRNA (Fig. 2D), suggesting that receptor Smad is required for the down-regulation of Cited2, and Smad2 or Smad3 may compensate each other in individual knockdown experiments.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
