Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response

The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms

Protective Effects of Epigallocatechin Gallate on Colon Preneoplastic Lesions Induced by 2-Amino-3-Methylimidazo[4,5-f ] Quinoline in Mice

Epigallocatechin gallate (EGCG), a key active ingredient in green tea, has multiple anticarcinogenic effects. The aim of the present study was to investigate if EGCG could prevent the formation of colon aberrant crypt foci (ACF) induced by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) and to explore possible mechanisms for resultant effects. Sixty male BALB/cA nude, immunodeficient mice were divided into six groups including a normal unexposed control, mice induced with IQ alone, three groups treated with varying doses of EGCG post–IQ induction, and a EGCG-treated control population. Six weeks later, the mice were killed, and tissues subjected to hematoxylin-eosin (H&E) and 0.2% methylene blue staining to observe histopathological alterations of colon mucus and the formation of ACF, respectively. Protein expression of NF-E2–related factor 2 (Nrf2) was assessed via immunohistochemistry (IHC) and Western analysis, and mRNA levels of Nrf2 and uridine 5′-diphosphate-glucuronosyltransferase (UGT)1A10 were determined in colon tissues. Our results demonstrate that, compared with IQ-induced controls, the degree of atypical hyperplasia decreased and the number of total ACF and total AC also decreased significantly (P < 0.05 and P < 0.01, respectively) in mice belonging to all EGCG dosing groups. At the same time, the protein levels of Nrf2 detected by IHC and Western blotting increased (both P < 0.01 compared with IQ group), and the mRNA levels of Nrf2 and UGT1A10 increased (both P < 0.01 compared with IQ group). In conclusion, EGCG had preventive effects on preneoplastic lesions induced by IQ. Our observations suggest that this effect may be the result of activation of the Nrf2-UGT1A10 signaling pathway