Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.
Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.
School of Nutrition, Chung Shan Medical University, Taichung 402, Taiwan.
Ai-Hsuan
AH
Lin
Haw-Wen
HW
Chen
Cheng-Tze
CT
Liu
Chia-Wen
CW
Tsai
Chong-Kuei
CK
Lii
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media; D015870:Gene Expression; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism*; D008297:Male; D008715:Methionine / Q000008:administration & dosage*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
D003470:Culture Media; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D008715:Methionine; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1