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

Oncogene volume 24, pages 3864–3874 (2005)Cite this article

Abstract

Transforming growth factor beta (TGF-β) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-β responsive genes. To determine if disruption of specific Smad protein–protein interactions would selectively inhibit responses to TGF-β 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-β responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-β-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-β-induced gene expression.

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Acknowledgements

We are grateful to Drs Joan Massagué, Peter ten Dijke, Rik Derynck and Liliana Attisano for Smad expression constructs and luciferase reporter constructs. We thank Dr Allen Comer for establishing the Smad baculovirus expression system, Angela Speer for technical assistance in the early stages of this project and Dr Norman Drinkwater for advice on statistical analysis and use of the MStat 4.01 program. We thank Kathy Schell, Joan Batchelder and Joey Puchalski in UWCCC Flow Cytometry Facility for their assistance on FACS sorting. This work was supported by R01 CA090875 to FMH, R21 DK064336 to Dr Bryan Becker and Cancer Center Support Grant P30 CA014520. QC was supported by the Cremer Foundation and BZ was supported by the pre-doctoral training grant in Cancer Biology T32-CA09135.

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  1. McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Qiqi Cui, Sang Kyun Lim, Bryan Zhao & Francis Michael Hoffmann

  2. Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Francis Michael Hoffmann

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Correspondence to Francis Michael Hoffmann.

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Cui, Q., Kyun Lim, S., Zhao, B. et al. Selective inhibition of TGF-β responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP. Oncogene 24, 3864–3874 (2005). https://doi.org/10.1038/sj.onc.1208556

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