Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3

J Biol Chem. 2003 Oct 31;278(44):43470-9. doi: 10.1074/jbc.M302339200. Epub 2003 Aug 13.


Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-gamma on alpha2(I) procollagen gene (COL1A2) transcription through the IFN-gamma response element located between -161 and -150. Here we report that YB-1 counter-represses TGF-beta-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around -265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-gamma-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-gamma in the absence and presence of TGF-beta. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-beta and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-gamma/YB-1 counteracts TGF-beta/Smad3. They also indicate that IFN-gamma/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-gamma response element and through a cross-talk with the TGF-beta/Smad signaling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Blotting, Western
  • CCAAT-Enhancer-Binding Proteins / metabolism*
  • Cells, Cultured
  • Collagen / metabolism
  • Collagen Type I
  • Cytoplasm / metabolism
  • DNA-Binding Proteins / metabolism*
  • Fibroblasts / metabolism
  • Gene Deletion
  • Glutathione Transferase / metabolism
  • Humans
  • Interferon-gamma / physiology*
  • Microscopy, Fluorescence
  • Models, Biological
  • NFI Transcription Factors
  • Nuclear Proteins
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Small Interfering / metabolism
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction*
  • Skin / metabolism
  • Smad3 Protein
  • Time Factors
  • Trans-Activators / metabolism*
  • Transcription Factors*
  • Transcriptional Activation
  • Transfection
  • Transforming Growth Factor beta / metabolism*
  • Y-Box-Binding Protein 1


  • CCAAT-Enhancer-Binding Proteins
  • Collagen Type I
  • DNA-Binding Proteins
  • NFI Transcription Factors
  • Nuclear Proteins
  • RNA, Small Interfering
  • Recombinant Proteins
  • SMAD3 protein, human
  • Smad3 Protein
  • Trans-Activators
  • Transcription Factors
  • Transforming Growth Factor beta
  • Y-Box-Binding Protein 1
  • YBX1 protein, human
  • Interferon-gamma
  • Collagen
  • Glutathione Transferase