NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis

Abstract

Background and aims: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1β), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC.

Approach and results: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r^-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r^-/- /DSS-PN mice. Knockdown of NF-κB in IL-1β-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1β, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC.

Conclusions: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

FIG. 1.

Effect of PNAC, LPS, and phytosterols on LRH-1 and its target gene expression. (A) Gene-expression analysis of hepatic Nr5a2/LRH-1 from Chow, DSS-Chow, PN, and DSS-PN treated mice. mRNA expression was determined after normalization to Hprt1 as an endogenous control gene and expressed relative to results obtained from untreated Chow controls. (B) Western analysis of LRH-1 and ABCG8 protein expression in liver homogenate from Chow, DSS-Chow, PN, and DSS-PN treated mice. (C) Quantification of integrated density values of the LRH-1 and ABCG8 immunoblot in (B). (D) Gene expression of NR5A2/LRH-1, ABCG5, ABCG8, and NR0B2/SHP in HepG2 cells from coculture experiments with human monocyte/macrophage THP-1 cells incubated with and without LPS for 4 hours and with or without stig+sito. Gene expression was determined after normalization to HPRT1 as an endogenous control gene and expressed relative to results obtained from untreated controls. (E) ChIP assay for LRH-1 binding to the promoter of Abcg5/8 in liver homogenate from chow, DSS-chow, PN, and DSS-PN mice. Data are presented as fold change over IgG. Statistical analysis was performed, and adjusted P values obtained using one-way ANOVA with Tukey’s correction for multiple comparisons (A,D) and by Student unpaired t test (C,E). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

FIG. 2.

IL-1β-induced NF-κB signaling suppresses LRH-1 and ABCG5 expression. (A) Gene-expression analysis of hepatic Nr5a2/LRH-1 from WT/chow, WT/DSS-PN, Il-1r^−/−/chow, and Il-1r ^−/−/DSS-PN mice. mRNA expression was determined after normalization to Hprt1 as an endogenous control gene and expressed relative to results obtained from chow controls. ****P < 0.0001 versus all other groups. (B) Hepatic expression of Abcg8 in chow, WT/DSS-PN, and Il-1r^−/−/DSS-PN mice. ^#P < 0.0001 for all other conditions. (C) ChIP assay for NF-κB binding to the LRH-1 promoter in liver homogenate from WT/chow, WT/DSS-PN, and Il-1r^−/−/DSS-PN mice. Data are presented as fold change over IgG control. Statistical analysis was by one-way ANOVA with Tukey’s correction for multiple comparisons. ****P < 0.0001. (D) Immunoblot of protein expression of total NF-κB-p65 and p-NF-κB-p65 in liver homogenates from chow, WT/DSS-PN, and Il-1r^−/−/DSS-PN mice. (E) Quantification of integrated density values of immunoblots of total NF-κB-p65 normalized to GRB2 endogenous control relative to chow control. ***P < 0.001 and ****P < 0.0001. Ratio of p-NF-κB-p65 to total normalized NF-κB. ***P < 0.001. (F) mRNA expression of NR5A2/LRH-1 in HepG2 cells transfected with NF-κB siRNA and nontargeting siRNA control for 48 hours followed by IL-1β treatment overnight. Gene expression was normalized to HPRT1 and expressed relative to results from untreated controls. *P < 0.05 and ^#P < 0.01 versus all other groups. mRNA expression of ABCG5 in HepG2 cells. Gene expression was normalized to HPRT1 and expressed relative to results from untreated controls. *P < 0.05 and ^#P < 0.01 versus all other groups. Abbreviation: GRB2, growth factor receptor bound protein 2.

FIG. 3.

Effect of inhibition of LRH-1 on FXR target genes in cultured cells and of LRH1 agonist treatment in DSS-PN mice. Cultured cells were incubated with LRH-1 inverse agonist ML179 for 4 hours followed by addition of GW4064 +/− stig+sito overnight, and mRNA expression was then analyzed. (A) NR0B2/SHP in HepG2 cells. ^#P < 0.0001 versus GW4064 and GW4064 + stig+sito. (B) ABCC2/MRP2 in HepG2 cells. ^#P < 0.05 versus all groups except stig+sito + GW4064. (C) Abcb11/BSEP in primary mouse hepatocytes (Mouse 1°). ^#P < 0.0001 versus GW4064 group. (D) Nr0b2/SHP in primary mouse hepatocytes. ^#P < 0.0001 versus all other groups. (E) ABCB11/BSEP in Huh7 cells. ^#P < 0.0001 versus all other groups. *P < 0.05 versus all groups except stig+sito + GW4064. ^$P < 0.01 versus all groups except ML179 + GW4064. Gene expression was normalized to HPRT1/Hprt1. (F) Serum AST, ALT, total serum bile acids, and total bilirubin in chow, DSS-PN, and DSS-PN /DLPC (LRH1 agonist)–treated mice. Statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05 and ****P < 0.0001.

FIG. 4.

Decreased expression of ABCG5/8 enhances the inhibitory effect of phytosterols on FXR target gene expression. HepG2 cells were transfected with ABCG8 siRNA or nontargeting siRNA for 24 hours followed by addition of +/− GW4064 or +/− stig+sito overnight, after which cells were harvested and mRNA analysis was performed. (A) ABCG8. ^#P < 0.0001 versus all groups except siRNA control. ^$P < 0.0001 versus all groups except untreated control. (B) NR0B2/SHP. ^#P < 0.05 versus all groups except ⁺ and ^$. ⁺P < 0.05 versus all groups except ⁺ and ^#. ^$P < 0.05 versus all groups except ^# and ⁺. ^&P < 0.05 versus all other groups. (C) HepG2 cells were incubated with IL-1β (to reduce ABCG5/8 expression) for 4 hours followed by GW4064 +/− stig+sito overnight, after which cells were harvested and mRNA analysis of NR0B2/SHP was performed. ^#P < 0.05 versus all groups except GW4064 + stig+sito + IL-1β. (D) Primary mouse hepatocytes (Mouse 1°) were incubated as in (C), and Nr0b2/SHP mRNA was analyzed. ^#P < 0.01 versus all groups except stig+sito + IL-1β + GW4064. (E) Abcb11 in primary mouse hepatocytes. ^#P < 0.001 versus all groups except GW4064 + stig+sito + IL-1β. (F) Huh7 cells were incubated as in (C), and ABCB11 mRNA was analyzed. ^#P < 0.0001 versus all other groups. ⁺P < 0.0001 versus all other groups. ^$P < 0.05 versus all other groups. ^&P < 0.05 versus all other groups. Gene expression was determined after normalization to HPRT1 relative to results obtained from untreated controls. For all of these experiments, statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

FIG. 5.

Inhibition of ABCG5/8 in HepG2 cells increases phytosterol accumulation and down-regulation of NR0B2/SHP. HepG2 cells were incubated with LXR antagonist GSK2033 for 4 hours followed by addition of +/− GW4064 or +/− stig+sito overnight; cells were harvested and mRNA analyzed. (A) NR0B2/SHP. ^#P < 0.05 versus all conditions except stig+sito + GSK2033+GW4064. (B) Immunoblot analysis and quantification of SHP protein in hepatic lysates extracted from HepG2 cells incubated as describe in (A). ^#P < 0.05 versus all groups except GW4064 + stig+sito. SHP protein was normalized to GRB2 and expressed relative to untreated control. (C) LC/MS quantification of stigmasterol concentrations in HepG2 cells treated as described in (A). ^#P < 0.0001 versus all other groups. ⁺P < 0.0001 versus all other groups. (D) LC/MS quantification of sitosterol in HepG2 cells treated as described in (A). ^#P < 0.0001 versus all other groups. *P < 0.05 versus all groups except untreated. (E) ChIP assay demonstrating FXR binding to the promoter of NR0B2/SHP in cell homogenate from HepG2 cells incubated as described in (A). Data are presented as fold change over IgG. For all of these experiments, statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Abbreviation: GRB2, growth factor receptor bound protein 2.

FIG. 6.

Proposed role of LRH-1 in PNAC pathogenesis. Intestinal injury, dysbiosis, and hyperpermeability caused by intestinal failure promote LPS (and other pathogen-associated molecular patterns) absorption into portal vein, subsequently recruiting and activating liver macrophages to generate IL-1β, which binds to IL-1 receptor on hepatocytes. This triggers activation of NF-κB, which binds to promoter of NR5A2/LRH-1 to inhibit its expression, as well as FXR and LXR target genes. This results in suppression of downstream ABCG5/8 transcription and subsequent hepatocyte accumulation of phytosterols infused in the PN solution, which further antagonize FXR signaling and thus down-regulating canalicular ABCB11/BSEP and ABCC2/MRP2, promoting retention of bile acids and bilirubin and culminating in cholestatic injury. Retained PN phytosterols may also directly activate hepatic macrophages and magnify cytokine production. Abbreviation: FXRE, farnesoid X receptor response element.