title: Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3.
abstract: 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.
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Konohana-ku, Osaka 554-8558, Japan. higashik2@sc.sumitomo-chem.co.jp
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Higashi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Inagaki
authors.forename: Ko
authors.initials: K
authors.lastname: Fujimori
authors.forename: Atsuhito
authors.initials: A
authors.lastname: Nakao
authors.forename: Hideo
authors.initials: H
authors.lastname: Kaneko
authors.forename: Iwao
authors.initials: I
authors.lastname: Nakatsuka
mesh_terms: D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D024042:Collagen Type I; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D007371:Interferon-gamma; D003094:Collagen; D005982:Glutathione Transferase