S-nitrosylation attenuates pregnane X receptor hyperactivity and acetaminophen-induced liver injury
Qi Cui, Tingting Jiang, Xinya Xie, Haodong Wang, Lei Qian, Yanyan Cheng, Qiang Li, Tingxu Lu, Qinyu Yao, Jia Liu, Baochang Lai, Chang Chen, Lei Xiao, Nanping Wang
Qi Cui, Tingting Jiang, Xinya Xie, Haodong Wang, Lei Qian, Yanyan Cheng, Qiang Li, Tingxu Lu, Qinyu Yao, Jia Liu, Baochang Lai, Chang Chen, Lei Xiao, Nanping Wang
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Research Article Cell biology Hepatology

S-nitrosylation attenuates pregnane X receptor hyperactivity and acetaminophen-induced liver injury

Abstract

Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the S-nitrosylation of PXR (SNO-PXR) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for S-nitrosylation (SNO) modification. In hepatocytes, SNO suppressed both agonist-induced (rifampicin and SR12813) and constitutively active PXR (VP-PXR, a human PXR fused to the minimal transactivator domain of the herpes virus transcription factor VP16) activations. Furthermore, in acetaminophen-overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR–/– mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of SNO-PXR. In conclusion, PXR is posttranslationally modified by SNO in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a target for therapeutical intervention.

Authors

Qi Cui, Tingting Jiang, Xinya Xie, Haodong Wang, Lei Qian, Yanyan Cheng, Qiang Li, Tingxu Lu, Qinyu Yao, Jia Liu, Baochang Lai, Chang Chen, Lei Xiao, Nanping Wang

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Figure 3

S-nitrosylation destabilized PXR protein via proteasome-dependent degradation.

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S-nitrosylation destabilized PXR protein via proteasome-dependent degrad...
(A) HepG2 cells were infected with Ad-PXR for 24 hours, or mouse primary hepatocytes were exposed to GSNO (0.5 mM) for indicated times. Nuclear PXR protein level was detected by using Western blotting (n = 3). (B) HepG2 cells were infected with Ad-PXR for 24 hours before being exposed to GSNO (0.5 mM, 12 hours) and were then subjected to immunofluorescence staining for PXR. Nuclei were counterstained with DAPI. Graph shows the mean fluorescence intensity (n = 6). Scale bars: 100 μm. (C) HepG2 cells or mouse primary hepatocytes were pretreated with CHX (30 μg/mL, 1 hour) before the exposure to GSNO for indicated times. Nuclear proteins were extracted and analyzed (n = 3). (D and E) HepG2 cells or mouse primary hepatocytes were pretreated with MG132 (10 μM, 1 hour) (D) or CQ (20 μM, 1 hour) (E) before the exposure to GSNO for indicated times. Nuclear proteins were extracted and analyzed (n = 3). (F) HepG2 cells or mouse primary hepatocytes were treated with GSNO (0.5 mM, 12 hours) and MG132 (10 μM, 6 hours) before harvesting. PXR was immunoprecipitated using anti-PXR antibody. The immunoprecipitates (IP) and whole-cell extracts were analyzed by Western blotting using an anti-Ub and anti-PXR antibody, respectively (n = 3). All data were expressed as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple-comparison test (A), 2-tailed Student’s t test (B and F), or multiple-comparison 2-way ANOVA with Bonferroni’s post hoc test (C, D, and E); *P < 0.05, **P < 0.01.