Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, 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 protein. bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-duanyu1800 had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-duanyu1800 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-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-duanyu1800 was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of to monomeric Smad protein and disruption of active Smad complexes.

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