Abstract
Using recombinant adenoviral vectors and a dominant negative mutant of HNF-4, we have examined the contribution of hepatocyte nuclear factor 4 (HNF-4) to endogenous apolipoprotein AI and CIII mRNA expression. Overexpression of HNF-4 leads to a 7.4-fold increase in apolipoprotein CIII expression, while infection with the dominant negative mutant of HNF-4 reduces the level of apolipoprotein CIII mRNA by 80%, demonstrating that endogenous HNF-4 is necessary for apolipoprotein CIII expression. Experiments using the hepatoma cell lines, HepG2 and Hep3B, indicate that HNF-4 is also involved in the regulation of apolipoprotein AI expression in these lines. However, the effect of HNF-4 on apolipoprotein AI expression is much more dramatic in cell lines derived from intestinal epithelium. Infection of the intestinal-derived cell line IEC-6 with the HNF-4 adenovirus resulted in a greater than 20-fold increase in the level of apolipoprotein AI mRNA. These results indicate that HNF-4 does regulate apolipoprotein AI and CIII mRNA expression and suggest that HNF-4 is critical for intestinal apolipoprotein AI expression.
Highlights
The apolipoproteins are lipid-binding polypeptides involved in the transport and metabolism of cholesterol, triglycerides, and phospholipids [1]
Dominant Negative Mutants of Hepatocyte nuclear factor 4 (HNF-4) —To examine the role of HNF-4 on apolipoprotein gene transcription, we constructed expression vectors coding for full-length cDNAs of the HNF4␣1 and HNF-4␣2 splice forms as well as a truncated, dominant negative form of HNF-4␣1 (Fig. 1A). ⌬111HNF-4␣1 contains a deletion of the amino-terminal 111 amino acids, which include the DNA-binding domain. ⌬111HNF4␣1 fails to display any detectable DNA binding activity and should act as a dominant negative of HNF-4-induced transcription due to dimerization with wild-type HNF-4
The HNF-4␣1 deletion mutant blocked the transfectioninduced promoter activity of either HNF-4␣1 or HNF-4␣2 (Fig. 1C). These results indicate that this DNA-binding domain mutant of HNF-4␣1 behaves as a dominant negative and that HNF-4␣1 and HNF-4␣2 can dimerize with each other. This result appeared to be specific for HNF-4-induced activity as cotransfection of ⌬111HNF-4␣1 had no effect on peroxisome proliferator-activated receptor ␣ or retinoid X receptor ␣-induced promoter activity
Summary
The apolipoproteins are lipid-binding polypeptides involved in the transport and metabolism of cholesterol, triglycerides, and phospholipids [1] These proteins regulate the structural characteristics of lipoprotein particles as well as their metabolism and uptake by cell surface receptors. HNF-4 binds DNA as a homodimer and does not appear to form heterodimers with several other intracellular receptor family members, including retinoid X receptor ␣, , ␥, retinoid acid receptor ␣, or thyroid hormone receptor ␣ [16]. Since the apoAI and apoCIII genes are closely linked and appear to share regulatory elements [17], we have constructed recombinant adenoviral vectors for the dominant negative mutants and wild-type HNF-4 that has allowed us to examine the effects of modulating HNF-4 transcriptional activity on the endogenous expression levels of the apoAI and apoCIII genes
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