Hepatic Forkhead Box Protein A3 Regulates ApoA-I (Apolipoprotein A-I) Expression, Cholesterol Efflux, and Atherogenesis

Abstract

Objective: To determine the role of hepatic FOXA3 (forkhead box A3) in lipid metabolism and atherosclerosis. Approach and Results: Hepatic FOXA3 expression was reduced in diabetic or high fat diet-fed mice or patients with nonalcoholic steatohepatitis. We then used adenoviruses to overexpress or knock down hepatic FOXA3 expression. Overexpression of FOXA3 in the liver increased hepatic ApoA-I (apolipoprotein A-I) expression, plasma HDL-C (high-density lipoprotein cholesterol) level, macrophage cholesterol efflux, and macrophage reverse cholesterol transport. In contrast, knockdown of hepatic FOXA3 expression had opposite effects. We further showed that FOXA3 directly bound to the promoter of the Apoa1 gene to regulate its transcription. Finally, AAV8 (adeno-associated virus serotype 8)-mediated overexpression of human FOXA3 in the hepatocytes of Apoe^-/- (apolipoprotein E-deficient) mice raised plasma HDL-C levels and significantly reduced atherosclerotic lesions.

Conclusions: Hepatocyte FOXA3 protects against atherosclerosis by inducing ApoA-I and macrophage reverse cholesterol transport.

Keywords: FOXA3; atherosclerosis; cholesterol efflux; liver; macrophages.

Figure 1.. Hepatic FOXA3 is reduced in HFD-fed or diabetic mice as well as NASH patients.

A-C, Hepatic mRNA levels in chow or high fat diet (HFD)-fed mice were quantified by qRT-PCR (A) (n=6 per group). Protein levels were determined by Western blotting (B) and then quantified (C). D-F, Hepatic levels of mRNA (D) or proteins (E, F) were determined in ob/ob or lean (control) mice (n=6 per group). G-I, Hepatic levels of mRNA (G) or proteins (H, I) were determined in db/db or lean (control) mice (n=6 per group). J-K, Hepatic levels of mRNA (J) or proteins (K, L) were determined in normal individuals or NASH patients (n=8 per group). Cd36 and Srebf1 serve as positive controls in A and D or G, respectively. * P<0.05, ** P<0.01, *** P<0.001

Figure 2.. Hepatic over-expression of FOXA3 increases HDL-c and ApoA-I levels.

C57BL/6J mice were i.v. injected with adenoviruses expressing human FOXA3 (Ad-hFOXA3) or control adenoviruses (Ad-Empty) (n=8 per group). After 7 days, mice were sacrificed. A, Plasma HDL-c and VLDL/LDL-c levels. B, Relative mRNA levels of hepatic genes involved in lipoprotein uptake, HDL biogenesis or inflammation. C and D, Hepatic protein levels were determined by Western blotting (C) and then quantified (D). E, FPLC analysis of plasma lipoprotein profile. F, Protein levels in individual HDL fractions were determined by Western blotting. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 3.. Knockdown of hepatic FOXA3 decreases HDL-c and ApoA-I levels.

C57BL/6J mice were i.v. injected with adenoviruses expressing shRNA against LacZ (Ad-shLacZ) or Foxa3 (Ad-shFoxa3) (n=8 mice per group). Mice were sacrificed 7 days later. A, Plasma HDL-c and VLDL/LDL-c levels. B, Plasma ALT and AST level. C and D, Hepatic levels of genes involved in lipoprotein uptake or HDL biogenesis (B) or inflammation (C). E and F, Hepatic protein levels were determined by Western blotting (E) and then quantified (F). G, FPLC analysis of plasma lipoprotein profile. H, Protein levels in individual HDL fractions were determined by Western blotting. * P < 0.05. ** P < 0.01, *** P < 0.001

Figure 4.. Hepatic FOXA3 regulates macrophage cholesterol efflux and reverse cholesterol transport.

C57BL/6J mice were i.v. injected with adenoviruses expressing FOXA3 or shRNA against Foxa3 or their control adenoviruses. A and B, Cholesterol efflux was performed using ApoB-depleted plasma (n=6–7 per group). C-E, Macrophage reverse cholesterol transport was performed in mice injected with Ad-Empty or Ad-FOXA3. At 48 h, [³H]tracers in the plasma (C), liver (D) or feces (E) were determined (n=7–8 per group). F-H, Macrophage reverse cholesterol transport was performed in mice injected with Ad-shLacZ or Ad-shFoxa3. At 48 h, [³H]tracers in the plasma (F), liver (G) or feces (H) were determined (n=7–8 per group). * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 5.. Apoa1 is a direct target gene of FOXA3.

A, Mouse primary hepatocytes were infected with Ad-Empty or Ad-FOXA3 for 24 h. mRNA levels were determined by real-time qPCR (n=3). B, Pulse-chase analysis of ApoA-I synthesis. Mouse primary hepatocytes were infected with Ad-Empty or Ad-FOXA3 for 24 h, pulsed with [³H]-Leucine for 2 h, and chased for 2 h. Radio-labeled ApoA-I in the media, cells and media plus cells (M+C) were determined. C, HepG2 cells were transfected with pGL3-basic or pGL3-Apoa1 luciferase-promoter constructs together with pCMV6 (control) or pCMV-FOXA3. After 36 h, relative luciferase units (RLU) were determined (n=5). D, The Apoa1 promoter has two putative FOXA3 binding sites. E, HepG2 cells were transfected with Apoa1 luciferase-promoter constructs containing wild-type or mutant FOXA3 binding site(s). After 36 h, RLU was determined (n=6). F, ChIP assay was performed by incubating IgG or FOXA3 antibodies with liver extracts of mice infected with Ad-Empty or Ad-FOXA3 (n=4). G, EMSA was carried out using 293A cell lysates overexpressing FOXA3 in the presence of oligos containing wild-type (WT) or mutant FOXA3 binding sites. For the competing study, the addition of an FOXA3 antibody disrupted the interaction between wild-type oligo and FOXA3 protein * P < 0.05, ** P < 0.01, *** P<0.001

Figure 6.. AAV-mediated over-expression of FOXA3 in hepatocytes induces ApoA-I expression and raises HDL-C levels in Apoe^−/− mice.

Apoe^−/− mice were i.v. injected with AAV8-ALB-Null or AAV8-ALB-FOXA3 and then fed a Western diet for 3 months (n=10–12 per group). A and B, Hepatic protein levels were analyzed by Western blotting (A) and then quantified (B). C, Plasma HDL-c levels. D, Plasma VLDL/LDL-c levels. E and F, FPLC analysis of plasma cholesterol (chol) (E) or TG (F) lipoprotein profiles. In the inlet of E, the HDL fraction is shown. * P< 0.05, ** P< 0.01, *** P<0.001.

Figure 7.. Hepatocyte-specific over-expression of FOXA3 attenuates the development of atherosclerosis.

Apoe^−/− mice were i.v. injected with AAV8-ALB-Null or AAV8-ALB-FOXA3 and then fed a Western diet for 3 months (n=10–12 per group). A, Representative images of aortas. Red arrows point to lesions. B, Representative images of aortas stained by oil red O. C, En face lesion area (%) of aortas. D, Representative images of aortic roots stained by oil red O. E, Lesion size of aortic roots. F and G, Aortic roots were immunostained with an MOMA-2 antibody (F) and staining-positive areas were quantified (G). In D and F, scale bars stand for 200 μm. * P< 0.05, ** P< 0.01, *** P< 0.001