title: Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.
abstract: We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads.
authors.affiliation: Department of Basic Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology of Hellas, Heraklion GR-71110, Greece.
authors.forename: Wan-Chih
authors.initials: WC
authors.lastname: Chou
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Prokova
authors.forename: Keiko
authors.initials: K
authors.lastname: Shiraishi
authors.forename: Ulrich
authors.initials: U
authors.lastname: Valcourt
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
authors.forename: Margarita
authors.initials: M
authors.lastname: Hadzopoulou-Cladaras
authors.forename: Vassilis I
authors.initials: VI
authors.lastname: Zannis
authors.forename: Dimitris
authors.initials: D
authors.lastname: Kardassis
mesh_terms: D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D051838:COUP Transcription Factor I; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D051838:COUP Transcription Factor I; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; C497589:NR2F1 protein, human; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta