Smad7 Antagonizes TGF-β Signaling By Interfering With Functional Smad-DNA Complex Formation In The Nucleus

The transforming growth factor-beta (TGF-β) superfamily, which includes TGF-β, Nodal, bone morphogenetic protein (BMP), activin, inhibin and other related factors, have vital functions in controlling cell proliferation, differentiation and death during embryo development and tissue homeostasis. Altered signal transduction of these factors results in abnormal embryonic development and a variety of diseases. Therefore, the precise regulation of TGF-βsignaling is essential for its normal physiological functions. Smad7 plays an essential role in the negative feedback regulation of TGF-βsignaling. It has been shown to inhibit TGF-βsignaling at the receptor level by its stable interaction with the active type I receptors. Smad7 can interfere with the binding and thus activation of R-Smads to type I receptors, or recruiting E3 ubiquitin ligases Smurf1 and Smurf2 to receptors and thus targeting them for degradation in the ubiquitin-dependent manner. Herein, we showed that Smad7 is predominantly localized in the nucleus of Hep3B cells. Smad7 represses TGF-βsignaling in the nucleus by competing with signaling Smads to bind to DNA and by recruiting transcriptional repressor HDAC.The targeted expression of Smad7 in the nucleus conferred a superior inhibitory effect on TGF-βsignaling as determined by reporter assay in mammalian cells and by analysis of its effect on zebrafish embryogenesis. A Smad7 mutant which cannot bind to Smurfs and stays in the nucleus still retained inhibitory activity. Furthermore, Smad7 greatly repressed Smad3/4- and Smad1/4-enhanced the reporter genes expression in TGF-βtype I receptor TβRI-deficient R1B/L17 cells as well as in Hep3B cells, indicating that Smad7 can function independently of type I receptor. Oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad responsive element via its MH2 domain, and the DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-βtarget gene, by chromatin immunoprecipitation. We also provided evidence that Smad7 disrupts the formation of the TGF-β-induced functional Smad-DNA complex. Furthermore, Smad7 possesses the transcriptional repression activity. We demonstrated that trichostatin A (TSA), an inhibitor of HDAC, partially rescued the repressive function of Smad7 in TGF-βsignaling. Moreover, Smad7 could interact with HDAC1 and HDAC3 using immunoprecipitation assay. These studies indicated that Smad7 recruited HDAC to represses TGF-βsignaling in the nuclues. These findings together suggest that Smad7 inhibits TGF-βsignaling in the nucleus in a novel mechanism.