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.
Cui
Qiqi
Q
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
Lim
Sang Kyun
SK
Zhao
Bryan
B
Hoffmann
Francis Michael
FM
D000595:Amino Acid Sequence; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins; D045744:Cell Line, Tumor; D003345:Corticosterone; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms; D051821:Lymphoid Enhancer-Binding Factor 1; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D016212:Transforming Growth Factor beta / Q000502:physiology*; D014981:Xenopus
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C510350:FOXH1 protein, human; D051858:Forkhead Transcription Factors; C491117:Foxh1 protein, mouse; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D005982:Glutathione Transferase; D003345:Corticosterone