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 2

S-nitrosylation suppressed the transactivatioal capacity of PXR.

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S-nitrosylation suppressed the transactivatioal capacity of PXR. (A) Hep...
(A) HepG2 cells were cotransfected with HA-PXR, PXRE-luciferase receptor, and β-gal plasmids. After 24-hour transfection, cells were pretreated with GSNO (0.5 mM, 4 hours) before the exposure to rifampicin (10 μM, 24 hours) or SR12813 (1 μM, 24 hours). The luciferase activities were measured and normalized to β-gal activity (n = 3). (B and C) After pretreatment with GSNO, HepG2 cells were stimulated with rifampicin or SR12813 for 24 hours. The mRNA levels of CYP3A4 (B) and SULT1A1 (C) were assessed by using qPCR (n = 4). (D and E) HepG2 cells were coinfected with Ad–VP-PXR together with Ad-tTA in the presence (Mock) or absence of Tc (1 μg/mL) for 24 hours and then exposed to GSNO (0.5 mM, 24 hours). The mRNA levels of CYP3A4 (D) and SULT1A1 (E) were assessed (n = 4). (F and G) After pretreatment with GSNO, HepG2 cells were stimulated with rifampicin or SR12813 for 24 hours. ChIP assays were performed with PXR antibody and primers flanking the PXRE motif in the CYP3A4 (F) and UGT1A1 (G) promoter region. The qPCR results were expressed as fold change compared with IgG control (n = 3). (H and I) HepG2 cells were coinfected with Ad–VP-PXR together with Ad-tTA in the presence (Mock) or absence of Tc (1 μg/mL) for 24 hours and then exposed to GSNO (0.5 mM, 24 hours). ChIP assays were performed with PXR antibody and primers flanking the PXRE motif in the CYP3A4 (H) and UGT1A1 (I) promoter region (n = 5). Data were expressed as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple-comparison test; *P < 0.05, **P < 0.01.