Selective inhibition of TGF-beta responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP.

Transforming growth factor beta (TGF-beta) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-beta responsive genes. To determine if disruption of specific Smad protein-protein interactions would selectively inhibit responses to TGF-beta or generally interfere with Smad-dependent signaling, we developed three Smad-binding peptide aptamers ...
by introducing Smad interaction motifs from Smad-binding proteins CBP, FoxH1 and Lef1 into the scaffold protein E. coli thioredoxin A (Trx). All three classes of aptamers bound to Smads by GST pulldown assays and co-immunoprecipitation from mammalian cells. Expression of the aptamers in HepG2 cells did not generally inhibit Smad-dependent signaling as evaluated using seven TGF-beta responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-beta-induced expression from a reporter dependent on the Smad-FoxH1 interaction, A3-lux, by 50%. Trx-xFoxH1b also partially inhibited two reporters not dependent on a Smad-FoxH1 interaction, 3TP-lux and Twntop, and endogenous PAI-1 expression. Trx-Lef1 aptamer only inhibited expression of the Smad-Lef1 responsive reporter gene TwnTop. The Trx-CBP aptamer had no significant effect on reporter gene expression. The results suggest that Smad-binding peptide aptamers can be developed to selectively inhibit TGF-beta-induced gene expression.
Mesh Terms:
Amino Acid Sequence, Animals, Carcinoma, Hepatocellular, Carrier Proteins, Cell Line, Tumor, Corticosterone, DNA-Binding Proteins, Forkhead Transcription Factors, Gene Expression Regulation, Neoplastic, Genes, Reporter, Glutathione Transferase, Humans, Liver Neoplasms, Lymphoid Enhancer-Binding Factor 1, Open Reading Frames, Polymerase Chain Reaction, Smad3 Protein, Trans-Activators, Transcription Factors, Transforming Growth Factor beta, Xenopus
Date: Jun. 02, 2005
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