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
View: Text | PDF
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

×

Figure 4

Identification of the S-nitrosylated cysteine residues in the human PXR.

Options: View larger image (or click on image) Download as PowerPoint
Identification of the S-nitrosylated cysteine residues in the human PXR....
(A) HEK293 cells were transfected with PXR WT or mutants (C182A, C307A) plasmids (24 hours) before the exposure to GSNO (0.5 mM, 4 hours). Cell lysates were subjected to IBP to detect S-nitrosylated PXR level (n = 3). (B) Transfected cells were treated with GSNO (0.5 mM, 12 hours) and MG132 for 6 hours before harvesting. PXR was immunoprecipitated using anti-PXR antibody. The IP and whole-cell extracts were analyzed by Western blotting using an anti-Ub and anti-PXR antibody, respectively (n = 4). (C) HEK293 cells were transfected with WT, C182A, or C307A plasmids for 24 hours and then treated with GSNO (0.5 mM, 12 hours). Nuclear PXR protein level was detected by using Western blotting (n = 3). (D) PXR WT, C182A, or C307A was cotransfected with PXRE-luciferase receptor into HEK293 cells. After 24 hours of transfection, cells were pretreated with GSNO (0.5 mM, 4 hours) before the exposure to SR12813 (1 μM, 24 hours). The luciferase activities were measured and normalized to β-gal activity (n = 4). (E) HEK293 cells were transfected with PXR WT, C182A, or C307A for 24 hours. After pretreatment with GSNO (0.5 mM, 4 hours), the cells were stimulated with SR12813 (1 μM, 24 hours). The mRNA level of CYP3A4 was assessed (n = 4). (F) ChIP assays were performed (n = 4). All 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.