Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling

The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.     

Repression of endogenous Smad7 by Ski

The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-beta (TGF-beta)-responsive promoter at a repressed, basal level. However, there have been no reports so far that it indeed acts on a natural promoter. We have previously cloned the human Smad7 promoter and shown that it contains the 8-base pair palindromic Smad-binding element (SBE) necessary for TGF-beta induction. In this report, we have characterized the negative regulation of Smad7 promoter basal activity by Ski. We show that Ski inhibits the Smad7 promoter basal activity in a SBE-dependent manner. Mutation of the SBE abrogates the inhibitory effect of Ski on the Smad7 promoter. Moreover, mutation of the SBE increases the Smad7 promoter basal activity. Using the chromatin immunoprecipitation assay, we further show that Ski together with Smad4 binds to the endogenous Smad7 promoter. Finally, we show that RNAi knockdown of Ski increases Smad7 reporter gene activity in transient transfection assays as well as elevating the endogenous level of Smad7 mRNA. Taken together, our results provide the first evidence that Ski is indeed a corepressor for Smad4, which can inhibit a natural TGF-beta responsive gene at the basal state.     

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.     

Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins

This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.     

Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation

Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism.     

Downregulation of Ski and SnoN co-repressors by anisomycin

Proteasome pathway regulates TGF-beta signaling; degradation of activated Smad2/3 and receptors turns TGF-beta signal off, while degradation of negative modulators such as Ski and SnoN maintains the signal. We have found that anisomycin is able to downregulate Ski and SnoN via proteasome as TGF-beta does, but through a mechanism independent of Smad activation. The mechanism used by anisomycin to downregulate Ski and SnoN is also independent of MAPK activation and protein synthesis inhibition. TGF-beta signal was the only pathway described causing Ski and SnoN degradation, thus this new effect of anisomycin on endogenous Ski and SnoN proteins suggests alternative processes to downregulate these negative modulators of TGF-beta signaling.