Human FOXA2 adenoviral particles

ATP-binding cassette transporter A1 (ABCA1) plays a key role in the biosynthesis of HDL by facilitating the efflux of cellular cholesterol and phospholipids to lipid-free apoA-I. Mutations in the ABCA1 gene cause Tangier disease, characterized by a near or complete absence of circulating plasma HDL. Here, researchers show that winged helix/forkhead box containing transcription factor A2 (FOXA2), previously shown to play a role in glucose and bile acid homeostasis in the liver and energy utilization in adipose tissue, is a negative regulator of ABCA1 gene expression in hepatocytes. The ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of these sites are located in a region of the ABCA1 promoter that is rich in transcriptional repressor protein binding elements, while the third site is in the core of the ABCA1 promoter TATA element. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or inhibition of FOXA2 gene expression by siRNA increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibits constitutive ABCA1 gene expression and induction of the ABCA1 gene by oxysterols and retinoids through the nuclear receptors LXRα/RXRα.

To evaluate the role of FOXA2 in ABCA1 gene expression in hepatocytes, the researchers used siRNA-mediated gene silencing. FOXA2-specific siRNA abolished endogenous FOXA2 gene expression in the human hepatoblastoma cell line HepG2 (bottom), and this silencing was associated with a significant increase in ABCA1 protein levels (top) (Figure 1a). Consistent with these findings, a 1.5-fold and 1.9-fold increase in the activity of the −688/+205 ABCA1 promoter was observed using two different concentrations of FOXA2-specific siRNA (75 and 150 nM), respectively, relative to the same concentration of control (scrambled) siRNA (Figure 1b). To further investigate the role of FOXA2 in ABCA1 gene regulation, the researchers generated recombinant adenovirus expressing FOXA2 and infected HepG2 cells with it. Upregulation of FOXA2 in HepG2 cells was associated with a significant 35% decrease in basal ABCA1 mRNA levels (Figure 1c). Western blot analysis of infected HEK293T cells showed a dose-dependent increase in adenovirus-mediated FOXA2 protein expression (Figure 1d), while indirect immunofluorescence of infected HepG2 cells confirmed that adenovirus-derived FOXA2, similar to endogenous FOXA2, was localized exclusively in the nucleus (Figure 1e). Together, these studies suggest that the hepatocyte-specific transcription factor FOXA2 plays a negative role in ABCA1 gene expression in hepatocytes.

Figure 1. FOXA2 is a negative regulator of ABCA1 gene transcription. (Thymiakou E, Kardassis D., 2014)