Abstract
INrf2:Nrf2 are sensors of chemical/radiation stress. Nrf2 dissociates from INrf2 in response to a stress and translocates in the nucleus. This leads to induction of a battery of antioxidant genes that protect cells. Nrf2 is then exported out and degraded. INrf2 functions as an adaptor of ubiquitin ligase for ubiquitination and degradation of Nrf2. Here we demonstrate the presence of a novel feedback autoregulatory loop between INrf2 and Nrf2 that controls cellular abundance of INrf2 and Nrf2. Nrf2 controls its own degradation by regulating expression and induction of the INrf2 gene. The antioxidant treatment of cells led to nuclear localization and stabilization of Nrf2 and induction of INrf2 gene expression. Mutagenesis, transfection, and chromatin immunoprecipitation assays identified an antioxidant-response element in the reverse strand of the proximal INrf2 promoter that binds to Nrf2 and regulates expression and antioxidant induction of the INrf2 gene. In addition, short interfering RNA inhibition or overexpression of Nrf2 led to a respective decrease and increase in INrf2 gene expression. These results implicated Nrf2 in the regulation of expression and induction of INrf2. The induction of INrf2 followed ubiquitination and degradation of Nrf2 and suppression of INrf2 gene expression. In conclusion, Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it.
Highlights
The ARE was first identified as cis-element in the upstream regulatory region of the GSTA2 gene [5] and was found in the promoters of detoxifying enzyme genes such as glutathione S-transferases [6], NAD(P)H:quinone oxidoreductases (NQOs) [7, 8], gastrointestinal glutathione peroxidase [9], and peroxiredoxin1 [10]
The results revealed that mutation of toma HepG2 cells demonstrated time- and concentration- the ARE-r1 element in 1.1-kb INrf2 promoter resulted in the dependent increase in INrf2 RNA in response to t-BHQ treat- significant reduction in basal expression and abrogation of ment (Fig. 1D)
The results showed that antioxidant treatment induced the expression of INrf2
Summary
The ARE was first identified as cis-element in the upstream regulatory region of the GSTA2 gene [5] and was found in the promoters of detoxifying enzyme genes such as glutathione S-transferases [6], NAD(P)H:quinone oxidoreductases (NQOs) [7, 8], gastrointestinal glutathione peroxidase [9], and peroxiredoxin1 [10]. Among the three protein factors, Nrf is most potent transcription factor in regulation of basal and induced expression of antioxidant enzyme genes [11]. Cysteine thiol groups of INrf were shown to function as sensors for oxidative stress that are modified by the chemical inducers, causing formation of disulfide bonds between cysteines of two INrf peptides. This results in conformational change that renders INrf unable to bind to Nrf2 [20, 21]. Stress-induced activation of the Nrf pathway in normal cells is tightly regulated and confers cytoprotection against oxidative and electrophilic stress and carcinogens. Nrf controls its own degradation by regulating INrf levels in the cells
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