The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.

Oxidative stress accelerates the pathogenesis of a number of chronic diseases including cancer growth and its metastasis. Transcription factor NF-E2-related factor-2 (Nrf2), which regulates the cellular defense system against oxidative stress, elicits essential protection against chemical-induced carcinogenic insults. We recently demonstrate that the systemic deletion of Nrf2 leads to an increased susceptibility to cancer metastasis, which is associated with aberrant reactive oxygen species (ROS) accumulation in myeloidderived suppressor cells (MDSC). However, it remains elusive whether cellular antioxidant defense system in the myeloid lineage cells plays indispensable roles for metastatic cancer progression. We herein found that myeloid lineage-specific Nrf2-deficient mice exhibited an increased susceptibility to pulmonary metastasis of the mouse Lewis lung carcinoma cells, and ROS level was more highly elevated in MDSCs of cancerbearing Nrf2-deficient mice. Similarly, myeloid lineage-specific deletion of selenocysteine-tRNA gene (Trsp), which is essential for synthesis of antioxidant selenoenzymes, resulted in increased number of metastatic nodules along with ROS accumulation in MDSCs of cancer-bearing mice. These results thus indicate that the antioxidant systems directed by Nrf2 and selenoenzymes contribute to the clearance of ROS in MDSCs, efficiently preventing cancer cell metastasis. Consistent with this notion, a synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im), a potent Nrf2 inducer, attenuated the ROS production in MDSCs, and thereafter reduced metastatic nodules. Taken together, this study provides compelling lines of evidence that Nrf2 inducer retains therapeutic efficacy against cancer cell metastasis. Cancer Prev Res; 7(8); 835-44. Ó2014 AACR.

Background/Aims. Antitumor necrosis factor antibodies and calcineurin inhibitors have shown good therapeutic efficacy for steroid-refractory ulcerative colitis (UC). Although some studies have compared the efficacy of infliximab (IFX) and cyclosporin A, there are no published studies comparing IFX and tacrolimus (Tac). This study aimed to compare therapeutic efficacies between IFX- and Tac-based strategies for steroid-refractory UC. Methods. Between July 2009 and August 2013, 95 patients with steroid-refractory UC received either IFX (n = 48) or Tac (n = 47) in our hospital. In the IFX group, the patients continued to receive maintenance treatment with IFX. In the Tac group, patients discontinued Tac treatment up to 3 months and subsequently received thiopurine. We retrospectively compared the therapeutic outcomes between the groups. Results. There was no significant difference in the colectomy-free rate, clinical remission rate, and clinical response rate at 2 months between the groups. However, relapse-free survival was significantly higher in the IFX group than in the Tac group (p < 0.001; log-rank test). The proportions of serious adverse events did not differ between the groups. Conclusion. The findings of our study showed that IFX and Tac have similar short-term therapeutic efficacy for steroid-refractory UC. Maintenance treatment with IFX, however, yields better long-term outcomes than Tac-thiopurine bridging treatment.