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-S MAD4 transactivated (15-70-fold) the -890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mu tant repressed the apoCIII promoter activity by 50%, suggesting that SMAD p roteins participate in apoCIII gene regulation. Transactivation required th e presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocy te nuclear factor-4 resulted in a strong synergistic transactivation of the -890/+24 apoCIII promoter, proximal promoter segments, or synthetic promot ers containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synt hesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediat ed transactivation. Co-immunoprecipitation and GST pull-down assays provide d evidence for physical interactions between SMAD3, SMAD4, and hepatic nucl ear factor-4, Our findings indicate that transforming growth factor beta an d its signal transducer SMAD proteins can modulate gene transcription by no vel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an impo rtant role in apolipoprotein gene expression and lipoprotein metabolism.