Fibroblast Growth Factor 11 Inhibits Hepatitis B Virus Gene Expression Through FXRα Suppression

Abstract

Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF family, which shows different signal transmission compared with other FGF superfamily members. The molecular function of FGF11 is not clearly understood. In this study, we identified the inhibitory effect of FGF11 on hepatitis B virus (HBV) gene expression through transcriptional suppression. FGF11 decreased the mRNA and protein expression of HBV genes in liver cells. While the nuclear receptor FXRα1 increased HBV promoter transactivation, FGF11 decreased the FXRα-mediated gene induction of the HBV promoter by the FXRα agonist. Reduced endogenous levels of FXRα by siRNA and the dominant negative mutant protein (aa 1-187 without ligand binding domain) of FXRα expression indicated that HBV gene suppression by FGF11 is dependent on FXRα inhibition. In addition, FGF11 interacts with FXRα protein and reduces FXRα protein stability. These results indicate that FGF11 inhibits HBV replicative expression through the liver cell-specific transcription factor, FXRα, and suppresses HBV promoter activity. Our findings may contribute to the establishment of better regimens for the treatment of chronic HBV infections by including FGF11 to alter the bile acid mediated FXR pathway.

Keywords: Antiviral effect; FGF11; FXR; HBx protein; Hepatitis B virus.

Fig. 1

FGF11 inhibits HBV promoter activity and gene expression of HBx. A Hep3B cells were co-transfected with the 1.3 × HBV-luc construct and the FGF11 expression plasmids in a dose-dependent manner. After 24 h of transfection, cell lysates were obtained, and luciferase activity was measured. Data shown are means ± SD of three independent experiments performed in duplicate. **p < 0.01, ***p < 0.001 compared with control. B Hep3B cells were transfected with the 1.2 mer HBV, pFlag-CMV2-FGF11 of human. At 24 h post-transfection, the total cellular RNA was extracted from the cells, and the levels of HBx and GAPDH were analyzed by real-time PCR. Data shown are means ± SD of three independent experiments performed in duplicate. **p < 0.01 compared with control. C Hep3B cells were transfected with 1 μg each of 1.2 mer 3 × flag HBV, pFlag-CMV2-FGF11 with the indicated combination. Whole cell lysates were analyzed for the expression of the indicated proteins by western blot with anti-flag antibody. Right panels indicate a relative fold of western blot results. ***p < 0.001 compared with control

Fig. 2

FGF11 suppresses FXRα-mediated gene activation of HBV promoter and HBx expression. A FXRα increases HBV promoter activation and FGF11 decreases HBV activation by FXRα. Hep3B, HepG2 and Chang cells were co-transfected with the 1.3 × HBV-luc construct, along with FXR and FGF11 expression plasmid as indicated combination. After 24 h of transfection, cell lysates were obtained, and luciferase activity was measured. Data shown are means ± SD of three independent experiments performed in duplicate. ***p < 0.001 compared with control. ^###p < 0.001 compared with FXRα1 group. B HepG2 cells were co-transfected with the 1.2 mer HBV, pCMV-FXR, pFlag-CMV2-FGF11 with the indicated combination. At 24 h post-transfection, the total cellular RNA was extracted from the cells, and the levels of HBx, FXRα, FGF11 and GAPDH were analyzed by real-time PCR. Data shown are means ± SD of three independent experiments performed in duplicate. *p < 0.05 compared with control. ^#p < 0.05 compared with FXRα1 group. C The HepG2 cells were co-transfected with the 1.2 mer 3 × flag HBV, pCMV-FXR, pFlag-CMV2-FGF11 of human with the indicated combination. Western blotting was performed on the cell extracts using anti-flag and anti-FXR serum. The equivalence of protein loading in the lanes was verified using anti-actin serum. Right panels indicate a relative fold of western blot results. *p < 0.05 compared with control. ^#p < 0.05 compared with FXRα1 group

Fig. 3

Agonists of FXRα largely increase HBV gene expression, but FGF11 inhibits FXRα agonist-derived HBV gene induction. A Hep3B, HepG2, and Chang cells were co-transfected with the 1.3 × HBV-luc construct, the FXR and FGF11 expression plasmid, and then maintained either under control conditions or in the presence GW4064 (10 μM) for 24 h. Data shown are means ± SD of three independent experiments performed in duplicate. **p < 0.01, ***p < 0.001 compared with control. ^###p < 0.001 compared with GW4064 group. ^$$p < 0.05 compared FXR versus FXR + GW4064. B, C The Hep3B cells were co-transfected with the 1.2 mer 3 × flag HBV construct and the FGF11 expression plasmid and maintained either under control conditions or in the presence of GW4064 10 μM (B) or CDCA 100 μM (C) for 24 h. Western blotting was performed on the cell extracts using anti-flag and anti-FXR serum. The equivalence of protein loading in the lanes was verified using anti-actin serum. Right panels indicate a relative fold of western blot results. ^#p < 0.05, ^##p < 0.01 compared with GW4064 group. D Hep3B, HepG2, and Chang cells were co-transfected with the 1.3 × HBV-luc construct, the FXR and FGF11 expression plasmid, and then maintained either under control conditions or in the presence of z-guggulsterone (10 μM) for 24 h. Data shown are means ± SD of three independent experiments performed in duplicate. **p < 0.01, ***p < 0.001 compared with control. ^###p < 0.001 compared with z-guggulsterone group. ^$$p < 0.05 compared FXR versus FXR + z-guggulsterone. E The Hep3B cells were co-transfected with the 1.2 mer 3 × flag HBV construct and the FGF11 expression plasmid and maintained either under control conditions or in the presence of z-guggulsterone (10 μM) for 24 h. Western blotting was performed on the cell extracts using anti-flag and anti-FXR serum. The equivalence of protein loading in the lanes was verified using anti-actin serum. Right panels indicate a relative fold of western blot results

Fig. 4

Reduced expression of FXRα selectively suppresses FGF11-mediated HBV inhibition. A FXRα siRNA counteracts FGF11-mediated HBx RNA transcription. Hep3B and Chang cells were transfected with pFlag-CMV2-FGF11 expression plasmids. At 24 h post-transfection, the total cellular RNA was extracted from the cells, and the levels of FGF11 and GAPDH were analyzed by RT-PCR (left) and quantitative real-time PCR (right). Data shown are means ± SD of three independent experiments performed in duplicate. ***p < 0.001 compared with control. ^#p < 0.05 compared with FGF11 group. B FXRα siRNA decreases HBx expression. Hep3B cells were transfected with pFlag-CMV2-FGF11 expression plasmids. Western blotting was performed on the cell extracts using anti-flag serum. The equivalence of protein loading in the lanes was verified using anti-actin serum. C Dominant negative mutant (LBD deletion) expression of FXRα strengthens FGF11-mediated HBx reduction. Hep3B cells were transfected with pFlag-CMV2-FGF11 and HA-FXRα1(LBD-) expression plasmids. Western blotting was performed on the cell extracts using anti-flag and HA antibodies

Fig. 5

FGF11 decreases FXRα protein stability. A Ectopic FGF11 expression decreases cellular protein level of FXRα. Hep3B cells were transfected with pFlag-CMV2-FGF11 expression plasmids. After 24 h of transfection, western blotting was performed on the cell extracts using anti-FXRα antibody. B FGF11 expression decreases de novo protein synthesis of FXRα1. Hep3B cells expressing FXRα and flag-FGF11 were treated with 10 μg/ml CHX or 25 μM MG-132 for the indicated period. Whole cell lysates were analyzed for the expression of the FGF11 proteins by western blot

Fig. 6

FGF11 interacts with FXRα through the N-terminal domain of FXRα. A FGF11 interacts with intact FXRα protein in the co-immunoprecipitation. B, C The N-terminal domain of FXRα is required for protein–protein interaction with FGF11. At 24 h post-transfection, cell lysates were collected for co-immunoprecipitation with beads conjugated with HA antibody (B) and flag antibody (C). The bound proteins were detected by western blot with flag antibody (B) and HA antibody (C)

Fig. 7

Inhibitory effect of FGF11 on HBV replication in in vivo mouse model. 10 μg of 1.2 mer HBV replicon and 1 × 10¹¹ pfu of adenoviruses were injected into the tail veins of C57Bl/6J mice. Quantitative real-time PCR detection of HBV DNA in liver tissue at day 7 after HI. Data shown are means ± SD of independent experiments performed in duplicate. **p < 0.01 compared with control