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Vol. 17, Issue 9, 3819-3831, September 2006

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Inhibition of Transforming Growth Factor-1–induced Signaling and Epithelial-to-Mesenchymal Transition by the Smad-binding Peptide Aptamer Trx-SARA

Bryan M. Zhao^*, and F. Michael Hoffmann^*,

*McArdle Laboratory for Cancer Research and Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706

Submitted October 31, 2005; Revised May 10, 2006; Accepted June 7, 2006
Monitoring Editor: Carl-Henrik Heldin

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor (TGF-) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and -catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-–induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Address correspondence to: F. Michael Hoffmann ( fmhoffma{at}wisc.edu)

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