SMAD Proteins Transactivate the Human ApoCIII Promoter by Interacting Physically and Functionally with Hepatocyte Nuclear Factor 4

Abstract

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 ␤ 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.

Identification of Novel TGF-β/Smad Gene Targets in Dermal Fibroblasts using a Combined cDNA Microarray/Promoter Transactivation Approach

“…Interestingly, when a 2.5-kilobase pair COL6A2 promoter fragment (24) was tested in an identical experimental system, we observed that it did not confer either TGF-␤ or Smad3 responsiveness (not shown), suggesting a differential regulation of the three genes encoding type VI collagen by TGF-␤, where both COL6A1 and COL6A3 are coordinately regulated and are direct Smad targets, whereas COL6A2 is not. These data differ slightly from previous observations indicating specific up-regulation of COL6A3 but not COL6A1 or COL6A2 by TGF-␤, when mRNA steady-state levels were detected after 48 h of stimulation (46).…”

“…Specifically, at present, only about a dozen genes are known to contain Smad-responsive regions, binding Smad complexes directly or indirectly. These genes include, in order of characterization as Smad targets, type VII collagen (COL7A1) (13), plasminogen activator inhibitor-1 (14), the cyclin-dependent kinase inhibitor p21 (15), JunB (16), and more recently, COL1A2 (17), c-Jun (18), immunoglobulin germline C␣ (19), Smad7 (20), human germline IgA (21), plateletderived growth factor-␤ (22), ␤5 integrin (23), apoCIII (24), and the cyclin-dependent kinase inhibitor p15 (25). Interestingly, to date, all Smad gene targets identified downstream of TGF-␤ are Smad3-, not Smad2-, dependent.…”

Orphan Nuclear Receptor Small Heterodimer Partner Inhibits Transforming Growth Factor-β Signaling by Repressing SMAD3 Transactivation

“…Moreover, Smad3 and Smad4, but not Smad2, physically interact with HNF-4 via their MH1 domains. TGF-␤-regulated Smad3 and Smad4 transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone-response element that binds HNF-4 (50,51), and retinoic acid receptors interfere with Smad signaling in a ligand-dependent manner (52). PIASy, an inhibitor of activated STAT, can repress Smad transcriptional activity by interacting with histone deacetylase (53), and interferon-␥ can interfere with TGF-␤ signaling through a direct interaction between YB-1, the Y box-binding protein, and Smad3, which interferes with the interaction between Smad3 and p300 (54).…”

Critical Role of Residues Defining the Ligand Binding Pocket in Hepatocyte Nuclear Factor-4α

“…that binds HNF-4␣ (13). This transcriptional cross-talk between the TGF-␤-regulated Smads and HNF-4␣ may play an important role in various hepatocyte functions that are regulated by TGF-␤ and Smads (14).…”