SMAD proteins transactivate the human ApoCIII promoter by interacting physically and functionally with hepatocyte nuclear factor 4.
Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15-70-fold) the -890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the -890/+24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor beta and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.
Kardassis
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Department of Basic Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, F.O.R.T.H., Heraklion GR-71110, Crete, Greece.
Pardali
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K
Zannis
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D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics*; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000502:physiology*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D010750:Phosphoproteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020218:Response Elements; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000502:physiology; D014407:Tumor Cells, Cultured
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; 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