Farnesoid X receptor represses hepatic human APOA gene expression

Abstract

High plasma concentrations of lipoprotein(a) [Lp(a), which is encoded by the APOA gene] increase an individual's risk of developing diseases, such as coronary artery diseases, restenosis, and stroke. Unfortunately, increased Lp(a) levels are minimally influenced by dietary changes or drug treatment. Further, the development of Lp(a)-specific medications has been hampered by limited knowledge of Lp(a) metabolism. In this study, we identified patients suffering from biliary obstructions with very low plasma Lp(a) concentrations that rise substantially after surgical intervention. Consistent with this, common bile duct ligation in mice transgenic for human APOA (tg-APOA mice) lowered plasma concentrations and hepatic expression of APOA. To test whether farnesoid X receptor (FXR), which is activated by bile acids, was responsible for the low plasma Lp(a) levels in cholestatic patients and mice, we treated tg-APOA and tg-APOA/Fxr-/- mice with cholic acid. FXR activation markedly reduced plasma concentrations and hepatic expression of human APOA in tg-APOA mice but not in tg-APOA/Fxr-/- mice. Incubation of primary hepatocytes from tg-APOA mice with bile acids dose dependently downregulated APOA expression. Further analysis determined that the direct repeat 1 element between nucleotides -826 and -814 of the APOA promoter functioned as a negative FXR response element. This motif is also bound by hepatocyte nuclear factor 4α (HNF4α), which promotes APOA transcription, and FXR was shown to compete with HNF4α for binding to this motif. These findings may have important implications in the development of Lp(a)-lowering medications.

Figure 1. Low plasma Lp(a) levels in patients with obstructive jaundice.

Plasma samples from 20 patients suffering from obstructive jaundice of different etiology were assayed for (A) total bile acids and (B) Lp(a), before and after surgical or endoscopic treatment. Values are expressed as mean ± SEM. See Supplemental Table 1 for details.

Figure 2. Drastic reduction in plasma levels and hepatic mRNA expression of APOA in a mouse model of cholestasis.

tg-APOA mice were subjected to biliary obstruction by CBDL (n = 3 per group) or sham operation (n = 4 per group) for 3 days. (A and B) Total bile acids and bilirubin were measured in serum. Data are presented as mean ± SD. ***P ≤ 0.001, when compared with sham-operated mice. (C) Plasma levels of APOA were measured by DELFIA and are expressed as mean ± SD (***P ≤ 0.001). (D) Liver APOA mRNA levels were analyzed by real-time quantitative PCR normalized to cyclophilin and are expressed relative to those of sham-operated mice. Results represent mean ± SEM (***P ≤ 0.001).

Figure 3. CA decreases plasma levels and hepatic expression of APOA in tg-APOA mice but not in tg-APOA/Fxr^–/– mice.

tg-APOA mice (n = 8 per group) and tg-APOA/Fxr^–/– mice (n = 8 per group) expressing human APOA were fed 0.2% CA (w/w) mixed in normal chow for 5 days. Control mice received normal rodent chow. (A and D) Plasma levels of APOA were measured by DELFIA and are expressed as mean ± SD (**P ≤ 0.01). (B and E) Mouse liver APOA mRNA levels were analyzed by real-time quantitative PCR and normalized to cyclophilin and are expressed relative to those of control mice. Results represent mean ± SEM (***P ≤ 0.001). (C and F) Western blot analysis and densitometric quantification of APOA levels in the protein extracts from liver tissue (expressed as mean ± SD relative to controls; **P ≤ 0.01). The gene expression profile was analyzed in (G) tg-APOA mice and (H) tg-APOA/Fxr^–/– mice by real-time quantitative PCR. mRNA expression in control mice was arbitrarily set to 1 and normalized to that of cyclophilin. Results represent mean ± SEM (***P ≤ 0.001, **P ≤ 0.01, *P < 0.05).

Figure 4. FXR agonists downregulate APOA gene expression in a dose- and time-dependent manner in primary mouse hepatocytes.

(A) Primary mouse hepatocytes from tg-APOA mice were incubated with increasing concentrations of CA (50, 100, and 200 μM) or vehicle (control) for 24 hours. APOA mRNA levels were analyzed by real-time quantitative PCR. These data are presented as mean ± SEM (**P ≤ 0.01, *P < 0.05). (B) Primary mouse hepatocytes were incubated with CA (200 μM) or vehicle for 12, 24, and 48 hours. APOA mRNA levels were measured by real-time quantitative PCR. Results represent mean ± SEM of 3 independent experiments (**P ≤ 0.01). (C) Western blotting and densitometric analyses of APOA expression in whole cell lysates from hepatocytes treated for 24 hours with increasing concentrations of CA (expressed as mean ± SD relative to controls; *P < 0.05). (D) Primary hepatocytes were treated with GW4064 (5 μM) for 24 hours and analyzed for APOA mRNA levels by real-time quantitative PCR. These data are presented as mean ± SEM of 3 independent experiments (**P ≤ 0.01). (E) Western blotting and densitometric analyses of APOA expression in whole cell lysates from hepatocytes treated for 24 hours with GW4064 (5 μM) (expressed as mean ± SD relative to controls; **P ≤ 0.01).

Figure 5. Bile acids and the nonsteroidal FXR agonist GW4064 downregulate human APOA promoter activity via FXR.

(A) Scheme of the full-length hAPOA –1,952/+52 promoter–driven luciferase reporter system. (B and C) HepG2 cells were transfected with the hAPOA –1,952/+52 promoter reporter plasmid (150 ng) in the presence of either the pcDNA3 (control) or FXR expression vector (150 ng). Cells were subsequently treated with CDCA (100 μM), GW4064 (500 nM), or vehicle for 36 hours. Values are normalized to internal control β-galactosidase and expressed as percentages. Transfections were performed in triplicates, and each experiment was repeated at least 3 times. (D) Scheme of the deletion constructs of the human APOA promoter used in the luciferase reporter assay. HepG2 cells were transfected with the indicated human APOA promoter reporter plasmids (150 ng) in the presence of pcDNA3 empty or FXR expression vector (150 ng). Cells were then treated with CDCA (100 μM) or vehicle for 36 hours. Values are normalized to internal control β-galactosidase activity. (E) Scheme showing wild-type and mutant sequences. Mutations are indicated in bold lowercase letters. Underlined letters define the DR-1 element. (F) Mutational analysis of the human APOA promoter. HepG2 cells were transfected with the wild-type and mutant (M1, M2) human APOA promoter reporter plasmids in the presence of pcDNA3 empty or FXR containing expression vector (150 ng). Cells were then treated with CDCA (100 μM) or vehicle for 36 hours. Values are normalized to β-galactosidase activity and expressed as percentages. Data are presented as mean ± SD (**P ≤ 0.01, *P < 0.05).

Figure 6. FXR binds to the DR-1 element of the human APOA promoter as a monomer.

(A) EMSAs were performed with radiolabeled IR-1 consensus FXRE (lanes 1–4), DR-1 WT (lanes 5–8), and DR-1 M2 (lanes 9–12) probes using in vitro transcribed/translated RXR (lanes 2, 6, and 10), FXR (lanes 3, 7, and 11), both RXR and FXR (lanes 4, 8, and 12), or unprogrammed reticulocyte lysate (lanes 1, 5, and 9) as indicated. (B) Competition EMSAs on radiolabeled DR-1 WT probe were performed by adding 50-fold, 100-fold, 200-fold molar excess of the indicated cold DR-1 WT (lanes 3–5) and 50-fold molar excess of cold DR-1 M2 (lane 6) and IR-1 (lane 7) probes. Numbering indicates relative intensity of the bands.

Figure 7. Effects of hepatocyte nuclear factor HNF4α overexpression on human APOA.

(A) Primary mouse hepatocytes were infected with either adenovirus coding for β-galactosidase (Ad-LacZ) or human HNF4A (Ad-HNF4α). Total RNA was extracted, and gene expression was measured by real-time quantitative PCR. Data represent mean ± SEM. (**P ≤ 0.01). (B) HepG2 cells were transfected with the hAPOA –1,952/+52 reporter plasmid (150 ng) in the presence of increasing amounts of HNF4α expression vector. Values represent mean ± SD (**P ≤ 0.01). (C) HepG2 cells were transfected with the hAPOA –1,952/+52 reporter plasmid in the presence or absence of FXR and HNF4α. Cells were then treated with CDCA (100 μM) or vehicle for 36 hours. Values are normalized to internal control β-galactosidase and expressed as percentages. Values represent mean ± SD (**P ≤ 0.01, *P < 0.05). (D) HNF4α binds to the DR-1 motif in the human APOA promoter. EMSAs with end-labeled DR-1 WT probe using in vitro transcribed/translated HNF4α (lanes 2). Competition analysis was performed by adding 50-fold (lane 3) and 100-fold (lane 4) molar excess of the indicated cold DR-1 WT probe. Underlined letters indicate the DR-1 element. (E) tg-APOA mice were fed normal chow or 0.2% CA chow for 24 hours, and livers were collected for ChIP analyses. For ChIP assay, sheared chromatin was immunoprecipitated with the indicated antibodies. The final DNA extractions were amplified by PCR using primer pairs covering the distal region and the DR-1 motif of the APOA gene promoter. As a positive control for FXR/RXR binding, the Shp gene promoter was amplified by PCR.