Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, SHP) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide in vitro, we demonstrated that the suppression of Shp expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun-mediated transcriptional repression of Shp Interestingly, in vivo induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C-C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH.

Keywords: chemokine; fibrosis; inflammation; liver injury; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; nuclear receptor; small heterodimer partner.

Figure 1.

Decrease of SHP in the livers of human NASH compared with NAFL. A, human liver specimens were obtained from the University of Kansas Liver Center, including 12 normal, 12 NAFL, and 8 NASH specimens. Left, representative images of liver sections stained with H&E or Picrosirius red from patients with NAFL or NASH. Original magnification, ×10. Right, relative level of SHP mRNA was determined by qPCR. *, p < 0.05. B, analysis of SHP mRNA expression in a microarray data set GSE48452. The number of specimens in each group was as follows: normal (n = 14), steatosis (n = 14), and NASH (n = 18). Data are represented as mean ± S.D. *, p < 0.05. C, left, Western blot analysis of SHP in human livers. Right, band intensities were calculated using ImageJ software. The relative expression of SHP was normalized to the expression of the loading control, β-actin. Data are presented as -fold change relative to that of the control. NS, no significance. *, p < 0.05.

Figure 2.

A mouse model carries NAFL progression to NASH. Two-month-old C57BL/6J male mice were fed a chow or HFCF diet for 1 and 5 months. n = 5/group. *, p < 0.05 HFCF versus chow-fed. A, body weight change over time. B, liver weight and liver to body weight ratio. C, serum levels of ALT and AST. D, serum levels of glucose, cholesterol, and triglycerides. E, glucose tolerance test. F, representative images of liver sections stained with H&E, oil red O, TUNEL, F4/80, or Picrosirius Red. Original magnification, ×40. G, histology scores of steatosis, inflammation, cell death, and fibrosis. Data are presented as mean ± S.D. *, p < 0.05 versus respective controls.

Figure 3.

SHP level is decreased in the livers of mice NASH. A, qPCR analysis of relative mRNA levels of genes related to lipid metabolism, inflammation, and fibrosis in the livers of mice fed chow or HFCF for 1 and 5 moths. n = 5 mice/group. *, p < 0.05 HFCF versus chow-fed. B, serum level of CCL2 was measured by ELISA. n = 5 mice/group. *, p < 0.05 HFCF versus chow-fed. C, qPCR analysis of Shp mRNA level in the livers of mice fed chow or HFCF. n = 5 mice/group. *, p < 0.05 HFCF versus chow-fed. D, left, Western blot analysis of SHP protein in the livers of mice fed chow or HFCF for 5 months. SHP H-160 is a rabbit polyclonal antibody and SHP H-5 is a mouse mAb. Both antibodies recognize the epitope corresponding to amino acid 1–160 mapping at the N terminus of SHP protein. Right, band intensities were calculated using ImageJ software. The level of SHP was normalized to the expression of loading control β-actin, and -fold changes relative to that of the controls are plotted. n = 5 mice/group. *, p < 0.05 HFCF versus chow-fed. E, representative images of liver sections stained with H&E in mice fed chow or MCD diet for 1 month. Original magnification, ×40. F, qPCR analysis of gene expression in the livers of mice fed chow or MCD diet for 1 month. n = 5 mice/group. Data are presented as mean ± S.D. *, p < 0.05 versus respective controls.

Figure 4.

Activation of JNK induces c-Jun targeting of Shp promoter leading to Shp suppression. A, qPCR analysis of gene expression in primary hepatocytes (Hepa), HSC, and resident macrophage KC isolated from mouse liver. Data are represented as mean ± S.D. *, p < 0.05. B, qPCR analysis of Shp mRNA expression in mouse hepatocytes. Hepatocytes were incubated with 0.5 mm PA or 100 ng/ml LPS for 6 h in the presence or absence of various inhibitors such as JNK inhibitor SP600125 (50 μm), NF-κB inhibitor BAY 11-7082 (5 μm), and PI3K inhibitor LY294002 (50 μm). The relative expression of Shp is normalized to the expression of internal control HPRT1. The -fold changes relative to that of the controls are plotted and presented as mean ± S.D. *, p < 0.05. C, Western blot analysis in the livers of mice fed chow or HFCF for 1 and 5 months. D, diagram shows the location of the TRE (core sequence TGAGTCA) site on the Shp promoter/reporter (Shp-Luc) and Shp-Luc mutant with a mutated TRE site. Three LRH1 binding sites are close to the TRE site. E, left, AML12 cells were transfected with Shp-Luc or its mutant with or without various expression plasmids. Luciferase activities were determined at 24 h post-transfection. Right, AML12 cells were transfected with Shp-Luc or its mutant for 24 h followed by incubation with SP600125 (50 μm) for 6 h. Data are displayed as the ratio of firefly luminescence divided by Renilla luminescence and represented as mean ± S.D. for triplicate experiments/group. *, p < 0.05. F, ChIP assay to determine the enrichment of c-Jun to Shp promoter. Left, AML12 cells overexpressed with c-Jun were harvested at 24 h post-transfection. pcDNA served as a transfection control. Right, AML12 cells were incubated with BSA control or 0.5 mm PA with or without JNK inhibitor SP600125 (50 μm) for 6 h. The cross-linked chromatin was immunoprecipitated by an antibody against c-Jun. The enriched DNA was amplified by qPCR and normalized to the input. -Fold changes relative to that of the controls are plotted and represented as mean ± S.D. *, p < 0.05. G, the enrichment of LRH1 to Shp promoter was revealed by ChIP assay. Left, AML12 cells overexpressed with c-Jun were harvested at 24 h post-transfection. Right, AML12 cells were incubated with 0.5 mm PA or BSA control for 6 h. The cross-linked chromatin was immunoprecipitated by an antibody against LRH1. The enriched DNA was amplified by qPCR and normalized to the input. The -fold changes relative to that of the controls are plotted and represented as mean ± S.D. *, p < 0.05.

Figure 5.

Increasing hepatocyte SHP levels in steatotic liver does not change liver steatosis. Two-month-old male C57BL/6J mice were fed a HFCF diet for 1 month to develop liver steatosis followed by tail vein administration of AAV8-Tbg-FlagSHP or control vector AAV8-Tbg-GFP. The mice remained on the HFCF diet for an additional 3 months. A, left, schematic diagram showing experimental design. Right, qPCR analysis of Shp mRNA levels in the liver. n = 5 mice/group. *, p < 0.05 SHP versus GFP. B, left, body weight change over time; middle, liver weight; right, liver weight to body weight ratio. n = 5 mice/group. C, serum levels of cholesterol, triglycerides, ALT, and AST. n = 5 mice/group. *, p < 0.05 SHP versus GFP. D, left, GTT. Right, the area under the curve (AUC) of GTT was calculated. n = 5 mice/group. E, representative images of liver sections stained with H&E, immunohistochemistry staining (IHC) of FLAG-SHP, and oil red O. Original magnification, ×40. n = 5 mice/group. F, liver triglycerides and cholesterol content. n = 5 mice/group.

Figure 6.

Hepatocyte SHP overexpression attenuates liver inflammation and fibrosis. Two-month-old male C57BL/6J mice were fed a HFCF diet for 1 month to develop liver steatosis followed by tail vein administration of AAV8-Tbg-FlagSHP or control vector AAV8-Tbg-GFP. Mice were continued on the HFCF diet for an additional 3 months. A, representative images of liver sections stained with F4/80 and Picrosirius red. Original magnification, ×40. n = 5 mice/group. B, liver collagen content was determined by hydroxyproline assay. n = 5 mice/group. Data are represented as mean ± S.D.; *, p < 0.05 SHP versus GFP. C, relative mRNA levels of genes related to inflammation, fibrosis, and lipid metabolism in the liver were determined by qPCR. Data are represented as mean ± S.D. for 5 mice/group; *, p < 0.05 SHP versus GFP. D, left, Western blot analysis of cytosolic and nuclear proteins (Pt) in the liver. Middle and right, band intensities were calculated using ImageJ software, and the intensities relative to that of the control were plotted. Data are represented as mean ± S.D.; *, p < 0.05 SHP versus GFP.

Figure 7.

Deletion of Shp in hepatocytes increases CCL2 production leading to macrophage proinflammatory polarization. A, schematic diagram shows experimental design. Primary hepatocytes from Shp^flox/flox mice were infected with adenovirus expressing Cre recombinase (Ad-Cre) or vector control (Ad-Null). CM was collected at 24 h post-adenoviral vector infection and used for RAW cell treatment. B, left, relative Shp mRNA levels were determined by qPCR. Data are represented as mean ± S.D. for triplicate experiments/group; *, p < 0.05. Right, representative images of immunofluorescence (IF) staining of p65 in hepatocytes. C, left, qPCR analysis of relative Ccl2 mRNA levels in hepatocytes. Right, CCL2 protein level in CM measured by ELISA. Data are represented as mean ± S.D. for experiments/group; *p < 0.05. D, RAW cells were incubated with CM from hepatocyte culture for 6 h, and the relative expression of genes involved in inflammation was determined by qPCR. Data are represented as mean ± S.D. for triplicate experiments/group; *, p < 0.05. E, left, representative images of RAW cell migration. RAW cells were incubated with CM in the presence or absence of recombinant mouse CCL2 (40 ng/ml) or anti-mouse CCL2 antibody (500 ng/ml). Cell migration was assessed after incubation for 12 and 24 h, respectively. Right, quantitation of cell migration was determined by measuring the pixel density of crystal violet–stained cells using ImageJ software. Data are represented as mean ± S.D. for five fields/sample. *, p < 0.05. F, left, diagram shows the location of two IκB sites on the mouse Ccl2 promoter reporter (Ccl2-Luc). Right, AML12 cells were transfected with Ccl2-Luc with various expression plasmids. Luciferase activities were determined at 24 h post-plasmid transfection. Data are calculated as the ratio of firefly luminescence divided by Renilla luminescence and presented as mean ± S.D. for triplicate experiments/group. *, p < 0.05. G, AML12 cells were overexpressed with FLAG-SHP or HA-p65 and harvested at 24 h post-plasmid transfection. Immunoprecipitation (IP) followed by Western blotting (WB) was employed to detect protein-protein interactions between FLAG-SHP and HA-p65.

Figure 8.

Hepatic cascade JNK/SHP/NF-κB/CCL2 regulates liver inflammation in NAFLD. Schematic diagram illustrates a novel regulatory network in hepatocytes, consisting of JNK/SHP/NF-κB/CCL2, that regulates macrophage recruitment and inflammation initiation critical for NAFLD progression.