Proteins of the transforming growth factor β(TGFβ) superfamily regulate diverse cellular responses, including cell growth and differentiation. After TGFβ stimulation, receptor-associated Smads are phosphorylated and form a complex with the common mediator Smad4. Here, we report the cloning of SMIF, a ubiquitously expressed, Smad4-interacting transcriptional co-activator. SMIF forms a TGFβ/bone morphogenetic protein 4 (BMP4)-inducible complex with Smad4, but not with others Smads, and translocates to the nucleus in a TGFβ/BMP4-inducible and Smad4-dependent manner. SMIF possesses strong intrinsic TGFβ-inducible transcriptional activity, which is dependent on Smad4 in mammalian cells and requires p300/CBP. A point mutation in Smad4 abolished binding to SMIF and impaired its activity in transcriptional assays. Overexpression of wild-type SMIF enhanced expression of TGFβ/BMP regulated genes, whereas a dominant-negative SMIF mutant suppressed expression. Furthermore, dominant-negative SMIF is able to block TGFβ-induced growth inhibition. In a knockdown approach with morpholino-antisense oligonucleotides targeting zebrafish SMIF, severe but distinct phenotypic defects were observed in zebrafish embryos. Thus, we propose that SMIF is a crucial activator of TGFβ signalling.
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We thank B. Vogelstein and K.W. Kinzler for the HCT116 and HCT116-SMAD4−/− cell lines and for the FAST1 cDNA, J. Wrana and L. Attisano for the ARE-lux construct, H.F. Lodish for TGFβ-RII cDNA, C.H. Heldin for TGFβ-RI and BMP-RI (ALK4) constructs and the Mv1lu-R42 cell line, M. Kawabata for BMP-RII and Smad6 cDNAs, R. Derynck for Smad3 cDNA, X.F. Wang for mSmad5 cDNA, B. Hill for Msx2 mRNA fragment, F. Oswald and R. Schmid for E1A constructs, E.J. Stanbridge for the SW480.7 cell line and J. Massague for Smad1, Smad2 and 3TP-lux constructs. We also thank C. Miething, N. von Bubnoff and J. Metzger for helpful technical advice, J. Sänger for technical help and K. Götze for critical reading of the manuscript. K.N. Astrahantseff is acknowledged for sharing unpublished data. This work was supported by a grant to JD from the German José-Carreras Stiftung.
Figure S1 SMIF immunoblot (PDF 481 kb)
Figure S2 Experiments similar to Figure 5d and e were performed in Mv1Lu-R42 cells, which express endogenous Smad4.
Figure S3 HCT Smad4-/- cells were transfected with TGFβ receptor II (TRII) and constructs as decribed in Figure 5f, with or without Smad4.
Figure S4 SMIF-Δ(124-390) competes with wildtype F-SMIF for the binding to Smad4.
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Bai, R., Koester, C., Ouyang, T. et al. SMIF, a Smad4-interacting protein that functions as a co-activator in TGFβ signalling. Nat Cell Biol 4, 181–190 (2002). https://doi.org/10.1038/ncb753
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