Interferon- Interferes with Transforming Growth Factor- Signaling through Direct Interaction of YB-1 with Smad3*

Transforming growth factor(TGF) and interferon(IFN) 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 IFNon 2(I) procollagen gene (COL1A2) transcription through the IFNresponse element located between 161 and 150. Here we report that YB-1 counter-represses TGF-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-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by doublestranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFNin the absence and presence of TGF. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGFand 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 Smadbinding 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/YB-1 counteracts TGF/Smad3. They also indicate that IFN/YB-1 inhibits COL1A2 transcription by dual actions: via the IFNresponse element and through a cross-talk with the TGF/Smad signaling pathway.