br Positive regulation of gene expression by Nrf br Repressi

Positive regulation of gene corticotropin-releasing factor by Nrf2
Repression of Nrf2 by Keap1 Because many xenobiotic inducers of NQO1 and GST enzyme activities contain a thiol-reactive electrophilic moiety (i.e. are soft electrophiles), Paul Talalay and colleagues predicted in 1988 that they would be recognized within the cell through reaction with sulfhydryl groups in a ‘sensor’ protein [25]. Thus following the discovery that Nrf2 mediates basal and/or inducible expression of Nqo1 and Gsta1 in mice ∗26, 27, it seemed possible that Nrf2 activity would be regulated through a ‘sensor’ protein that contains unique thiol groups. Yeast two-hybrid screening enabled identification of Keap1 as a repressor protein that binds to the Nrf2-ECH homology (Neh)2 domain of Nrf2 [28∗], and confers instability on Nrf2 5, 29. In mammalian species, Keap1 contains about ten reactive Cys residues (i.e. with thiolate anion side-chain) because they are located adjacent to basic amino acids. Consistent with the hypothesis that Keap1 represents an electrophile ‘sensor’ protein, many agents that induce Nrf2-target genes form adducts with Cys-151, Cys-273, Cys-288, Cys-434 and Cys-613 in Keap1 ∗30, 31, 32, ∗33. Most importantly, the ability of inducing agents to modify these Cys residues is inversely associated with the ability of CRLKeap1 to ubiquitylate Nrf2 [34*], and manipulation of the basic environment around Cys-151 in Keap1 diminishes the ability of inducing agents such as tert-butyl hydroquinone (tBHQ) and sulforaphane (SFN) to stabilize Nrf2 protein [33∗]; see Figure 1 for structures of tBHQ and SFN. Using knock-in of mutant forms of Keap1 that retain substrate adaptor activity but lack individual electrophile sensors, it has been demonstrated that Cys-151, Cys-273 and Cys-288 recognize different inducers [35]. Besides forming adducts with xenobiotics, Cys-216 and Cys-613 in Keap1 can form an intra-molecular disulfide bridge following exposure to H2O2, and this is thought to contribute to activation of Nrf2 by reactive oxygen species (ROS) 36, 37. Importantly, inducing agents stimulate conformational changes in the complex formed between Keap1 and Nrf2 that block ubiquitylation of the transcription factor by CRLKeap1[38].
Control of Nrf2 via the combined actions of the PTEN−GSK-3 pathway and β-TrCP
Certain, but not all, inducers that activate Nrf2 antagonize both Keap1 and GSK-3 Evidence suggests that tBHQ may simultaneously inhibit the Keap1-directed ubiquitylation of Nrf2 and the phosphorylation of Nrf2 by GSK-3 [52]. It is not however clear whether other inducing agents antagonize both Keap1 and GSK-3. To examine this possibility, and test whether antagonism of GSK-3 entails activation of PI3K and PKB/Akt, we treated Keap1 and Keap1+/+ MEFs with tBHQ or diethyl maleate (DEM), with the latter chosen because it is a potent inducing agent that has been employed in gene expression profiling and chromatin immunoprecipitation combined with high-throughput DNA sequencing 4, ∗28. As anticipated, immunoblotting showed that the basal level of mRNAs for both Hmox1 and Nqo1 is higher in Keap1 than Keap1+/+ fibroblasts. Treatment of Keap1+/+ fibroblasts with tBHQ (Figure 2A) or DEM (Figure 2B) induced both Hmox1 and Nqo1. Whilst induction was less obvious in Keap1−/− than Keap1 fibroblasts, both tBHQ and DEM increased Hmox1 and Nqo1 mRNA in Keap1−/− MEFs. Interestingly, the PI3K and PKB/Akt inhibitors, LY294002 and MK-2206, diminished both basal and inducible Hmox1 and Nqo1 mRNA levels in both wild-type and mutant MEFs. By contrast, the GSK-3 inhibitor CT99021 increased basal levels of Hmox1 and Nqo1 mRNA in both Keap1+/+ and Keap1 MEFs, which is consistent with previous results using the GSK-3 inhibitors LiCl and PDZD-8 [8∗]. This is an interesting finding because, unlike typical inducing agents, CT99021 is not a soft electrophile. It is also notable that CT99021 did not significantly increase the expression of Hmox1 or Nqo1 in Keap1−/− MEFs treated with tBHQ above the elevated level stimulated by the inducing agent, but this was not the case for DEM.