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

Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c- myc expression in the absence of TGF-β signaling

“…TGF-␤ signaling induces a Smad-E2F4/5-p107 complex to bind to a specific region of the c-myc promoter, called the TIE, and repress c-myc transcription (5). TrxLef1D binds to Smad proteins and inhibits Smad-dependent transcription at the Twntop luciferase reporter gene (11), but it seemed unlikely that TrxLef1D was acting on the Smad complex at the TIE of the c-myc promoter, especially because its effects on c-myc were observed in the absence of TGF-␤ signaling. This led us to hypothesize that a Smad protein might positively regulate c-myc transcription in a complex with LEF/TCF in the absence of TGF-␤ signaling.…”

“…Smad proteins are able to regulate cell proliferation by repressing c-myc and inducing cyclin-dependent kinase inhibitors such as p15Ink4b and p21Cip1/Waf1 through direct binding to the promoters of each gene (5,19,20). c-myc was a particularly interesting candidate gene that might be affected by TrxLef1D, because both Smads and LEF/TCF directly bind to the c-myc promoter (5,6), and a known target of TrxLef1D is a Smad-LEF/TCF complex (11). To test whether c-myc expression might be changed by TrxLef1D expression, a reporter gene driven by the c-myc promoter was cotransfected in HepG2 cells with plasmids expressing either TrxLef1D or TrxGA.…”

“…We have generated small focused libraries of Smad-binding domains from various Smad-interacting proteins displayed on a thioredoxin A scaffold (Trx). Several of these peptide aptamers bind to Smad proteins and inhibit TGF-␤/Smad signaling selectively (11,12). Among them, TrxLef1D, generated by inserting the Smad-binding domain from LEF1 into the Trx scaffold, was able to bind Smad1, -2, -3, -4, and -7, as does full length LEF1 (13)(14)(15)(16)(17).…”

“…Among them, TrxLef1D, generated by inserting the Smad-binding domain from LEF1 into the Trx scaffold, was able to bind Smad1, -2, -3, -4, and -7, as does full length LEF1 (13)(14)(15)(16)(17). TrxLef1D specifically inhibited a Smad-LEF/TCF complex-dependent reporter, Twntop-lux, without interfering with TGF-␤ activation of other Smad-dependent reporters (11).…”

“…HepG2 cells stably expressing TrxLef1D were generated by retroviral infection and sorted for high level expression of GFP to verify the infection (H2-TrxLef1D cells). In addition, HepG 2 cells expressing TrxGA, which has a tandem repeat of gly-ala in the Trx scaffold and has been used as a negative control aptamer in previous assays (11,12), were also prepared by the same procedure (H2-TrxGA cells). When each cell type was cultured in the absence or presence of TGF-␤, the proliferation of both H2-TrxLef1D and H2-TrxGA cells was inhibited by TGF-␤ treatment (Fig.…”