Distinct Regulations of HO-1 Gene Expression for Stress Response and Substrate Induction.

Heme oxygenase 1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas HO-1 gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which HO-1 gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of HO-1 utilize similar or distinct regulatory pathways, we developed an HO-1-DsRed-knock-in reporter mouse in which the HO-1 gene is floxed by loxP sites and the DsRed gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1-DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1-DsRed expression in the whole body in vivo and ex vivo. HO-1 inducers upregulated HO-1-DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1-DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated HO-1 induction and NRF2-independent substrate-mediated HO-1 induction. Thus, the HO-1 gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.

Author Correction: Nrf2 contributes to the weight gain of mice during space travel.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nrf2 contributes to the weight gain of mice during space travel.

Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans.

Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus.

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.

Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

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.

Amelioration of inflammation and tissue damage in sickle cell model mice by Nrf2 activation.

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the ß-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells' hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage.

Keap1 inhibition attenuates glomerulosclerosis.

BACKGROUND: NFE2-related factor 2 (Nrf2) is a master regulatory transcription factor for antioxidant genes. Inhibition of its adaptor protein, Kelch-like ECH-associated protein 1 (Keap1), activates Nrf2. Podocyte injury triggers the progressive deterioration of glomerular damage toward glomerulosclerosis. We examined whether modulation of the Keap1-Nrf2 system has an impact on this process. METHODS: Nrf2 null-mutant (KO) and Keap1 hypomorphic knockdown (KD) mice were crossed with NEP25 mice, in which podocyte-specific injury can be induced by an immunotoxin. RESULTS: Thiobarbituric acid reactive substances, 8-hydroxydeoxyguanosine and phosphorylated JNK were increased in the injured NEP25 kidney. Real-time PCR revealed that Keap1 KD upregulated Nrf2 target genes, including Gclc, Gclm, Gstp1, Gstp2 and Nqo1 in the glomerulus. However, podocyte injury did not upregulate these genes in Keap1 wild-type mice, nor did it further increase the expression of those genes in Keap1 KD mice. Three weeks after the induction of podocyte injury, glomerulosclerosis was considerably more attenuated in Keap1 KD mice than in control mice (median sclerosis index, 0.27 versus 3.03, on a 0-4 scale). Keap1 KD mice also showed considerably preserved nephrin staining (median index, 6.76 versus 0.91, on a 0-8 scale) and decreased glomeruli containing desmin-positive injured podocytes (median percentage, 24.5% versus 85.8%), along with a decrease in mRNAs for Fn1, Tgfb1, Col4a4 and Col1a2. CONCLUSIONS: Thus, podocyte injury cannot effectively activate Nrf2, but Nrf2 activation by Keap1 knockdown attenuates glomerulosclerosis. These results indicate that the Nrf2-Keap1 system is a promising drug target for the treatment of chronic kidney diseases.