c‐Jun NH2‐Terminal Protein Kinase Phosphorylates the Nrf2‐ECH Homology 6 Domain of Nuclear Factor Erythroid 2–Related Factor 2 and Downregulates Cytoprotective Genes in Acetaminophen‐Induced Liver Injury in Mice

Abstract

BaCKgRoUND aND aIMS: Acetaminophen (APAP) overdose induces severe liver injury and hepatic failure. While the activation of c-Jun NH 2 -terminal kinase ( JNK) has been implicated as a mechanism in APAP-induced liver injury, the hepatic defense system controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in the mitigation of APAP toxicity. However, the link between the two signaling pathways in APAP-induced liver injury (AILI) remains unclear. appRoaCH aND ReSUltS: In this study, we demonstrated that the activation of JNK in mouse liver following exposure to APAP was correlated with the phosphorylation of Nrf2 and down-regulation of the antioxidant response element (ARE)-driven genes, NAD(P)H:quinone dehydrogenase 1, glutathione S-transferase α3, glutathione S-transferase M1, glutathione S-transferase M5, and aldo-keto reductase 1C. The JNK inhibitor, SP600125, or knockdown of JNK by infection of adenovirus expressing JNK small interfering RNA, ameliorated the APAP induced liver toxicity, and inhibited the phosphorylation of Nrf2 and down-regulation of detoxifying enzymes by stabilizing the transcription factor. Mechanistically, JNK antagonized Nrf2-and ARE-driven gene expression in a Kelch-like ECH-associated protein 1-independent manner. Biochemical analysis revealed that phosphorylated JNK (P-JNK) directly interacted with the Nrf2-ECH homology (Neh) 1 domain of Nrf2 and phosphorylated the serine-aspartateserine motif 1 (SDS1) region in the Neh6 domain of Nrf2.CoNClUSIoNS: Mass spectrometric analysis identified serine 335 in the SDS1 region of mNrf2 as the major phosphorylation site for modulation of Nrf2 ubiquitylation by P-JNK. This study demonstrates that Nrf2 is a target of P-JNK in AILI. Our finding may provide a strategy for the treatment of AILI. (Hepatology 2020;71:1787-1801. N -acetyl-p-aminophenol (APAP; acetaminophen) is a commonly used non-narcotic analgesic drug used to reduce fever and relieve

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“…This interaction allows the phosphorylation of Ser residues at Neh6 degron domain of NRF2. Thus, it was demonstrated that p-JNK phosphorylates Ser335 at DSGIS motif of NRF2, codifying its degradation through a KEAP1-independent mechanism [249].…”

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“…Furthermore, fullerenol C 60 (OH) 24 is involved in phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) kinase, and then the kinases, which are regulated by extracellular signals, as well as c-Jun-N-terminal kinases [ 135 ]. All mentioned kinases phosphorylate Nrf2 and enable its translocation into the cell nucleus [ 163 , 164 ].…”

Reply:

“…First, our findings of the phosphorylation of nuclear erythroid 2 p45‐related factor 2 (Nrf2) and diminished mRNAs of NAD(P)H:quinone oxidoreductase 1 (Nqo1) , glutathione S‐transferase A3 ( Gstα3 ), glutathione S‐transferase M1 ( Gstm1 ), glutathione S‐transferase M5 ( Gstm5 ), and aldo‐keto reductase 1C ( AKR1C ) by 6 hours after acetaminophen (APAP) treatment (300 mg/kg) (Supporting Fig. S2B), ( 1 ) indicate that the Nrf2/Antioxidant responsive element (ARE) cytoprotective system is impaired from a very early time point. No significant changes in the protein levels were detected at the 6‐hour time point.…”