Emerging Role of GCN1 in Disease and Homeostasis.

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

Individual health-disease phase diagrams for disease prevention based on machine learning.

Early disease detection and prevention methods based on effective interventions are gaining attention worldwide. Progress in precision medicine has revealed that substantial heterogeneity exists in health data at the individual level and that complex health factors are involved in chronic disease development. Machine-learning techniques have enabled precise personal-level disease prediction by capturing individual differences in multivariate data. However, it is challenging to identify what aspects should be improved for disease prevention based on future disease-onset prediction because of the complex relationships among multiple biomarkers. Here, we present a health-disease phase diagram (HDPD) that represents an individual's health state by visualizing the future-onset boundary values of multiple biomarkers that fluctuate early in the disease progression process. In HDPDs, future-onset predictions are represented by perturbing multiple biomarker values while accounting for dependencies among variables. We constructed HDPDs for 11 diseases using longitudinal health checkup cohort data of 3,238 individuals, comprising 3,215 measurement items and genetic data. The improvement of biomarker values to the non-onset region in HDPD remarkably prevented future disease onset in 7 out of 11 diseases. HDPDs can represent individual physiological states in the onset process and be used as intervention goals for disease prevention.

Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.

Machine learning diagnosis by immunoglobulin N-glycan signatures for precision diagnosis of urological diseases.

Early diagnosis of urological diseases is often difficult due to the lack of specific biomarkers. More powerful and less invasive biomarkers that can be used simultaneously to identify urological diseases could improve patient outcomes. The aim of this study was to evaluate a urological disease-specific scoring system established with a machine learning (ML) approach using Ig N-glycan signatures. Immunoglobulin N-glycan signatures were analyzed by capillary electrophoresis from 1312 serum subjects with hormone-sensitive prostate cancer (n = 234), castration-resistant prostate cancer (n = 94), renal cell carcinoma (n = 100), upper urinary tract urothelial cancer (n = 105), bladder cancer (n = 176), germ cell tumors (n = 73), benign prostatic hyperplasia (n = 95), urosepsis (n = 145), and urinary tract infection (n = 21) as well as healthy volunteers (n = 269). Immunoglobulin N-glycan signature data were used in a supervised-ML model to establish a scoring system that gave the probability of the presence of a urological disease. Diagnostic performance was evaluated using the area under the receiver operating characteristic curve (AUC). The supervised-ML urologic disease-specific scores clearly discriminated the urological diseases (AUC 0.78-1.00) and found a distinct N-glycan pattern that contributed to detect each disease. Limitations included the retrospective and limited pathological information regarding urological diseases. The supervised-ML urological disease-specific scoring system based on Ig N-glycan signatures showed excellent diagnostic ability for nine urological diseases using a one-time serum collection and could be a promising approach for the diagnosis of urological diseases.

Association of mitochondrial DNA haplogroup and hearing impairment with aging in Japanese general population of the Iwaki Health Promotion Project.

Age-related hearing loss (ARHL) is a complex multifactorial disorder. Studies in animals, including mitochondria-mutator mice, and in human suggest that oxidative stress and mitochondrial disturbance play an important role in the pathoetiology of ARHL. Mitochondrial DNA (mtDNA) haplogroups are populations with genetically similar traits, and they have been reported to affect the mitochondrial function of oxidative phosphorylation. To gain further insights into the relationships between mitochondrial haplotypes and the susceptibility to cochlear aging, in this study, we aimed to elucidate how the differences in mtDNA haplogroups may affect ARHL development in Japanese general population. We focused on early onset ARHL, as the same mtDNA haplogroup can show either a negative or positive effect on systemic co-morbidities of ARHL that appear later in life. A total of 1167 participants of the Iwaki Health Promotion Project were surveyed in 2014, and 12 major haplotype groups (D4a, D4b, D5, G1, G2, M7a, M7b, A, B4, B5, N9, and F) were selected for the analysis. A total of 698 subjects aged 30 to 65 years were included in the statistical analysis, and the hearing loss group consisted of 112 males (40.3%) and 111 females (26.4%). Multiple logistic regression analysis showed that the male subjects belonging to haplogroup A had a significantly increased risk of hearing loss, whereas the female subjects belonging to haplogroup N9 had a significantly decreased risk of hearing loss. These results suggested that the mtDNA haplogroup may be an indicator for future risk of morbidity associated with ARHL.

Sulforaphane Increase Mitochondrial Biogenesis-Related Gene Expression in the Hippocampus and Suppresses Age-Related Cognitive Decline in Mice.

Sulforaphane (SFN) is a potent activator of the transcriptional factor, Nuclear Factor Erythroid 2 (NF-E2)-Related factor 2 (NRF2). SFN and its precursor, glucoraphanin (sulforaphane glucosinolate, SGS), have been shown to ameliorate cognitive function in clinical trials and in vivo studies. However, the effects of SGS on age-related cognitive decline in Senescence-Accelerated Mouse Prone 8 (SAMP8) is unknown. In this study, we determined the preventive potential of SGS on age-related cognitive decline. One-month old SAMP8 mice or control SAM resistance 1 (SAMR1) mice were fed an ad libitum diet with or without SGS-containing broccoli sprout powder (0.3% w/w SGS in diet) until 13 months of age. SGS significantly improved long-term memory in SAMP8 at 12 months of age. Interestingly, SGS increased hippocampal mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) and mitochondrial transcription factor A (TFAM), which are master regulators of mitochondrial biogenesis, both in SAMR1 and SAMP8 at 13 months of age. Furthermore, mRNAs for nuclear respiratory factor-1 (NRF-1) and mitochondrial DNA-encoded respiratory complex enzymes, but not mitochondrial DNA itself, were increased by SGS in SAMP8 mice. These results suggest that SGS prevents age-related cognitive decline by maintaining mitochondrial function in senescence-accelerated mice.

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage.

GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.

Role of Nrf2 in inflammatory response in lung of mice exposed to zinc oxide nanoparticles.

BACKGROUND: Zinc oxide nanoparticles (ZnO-NPs) are widely used in many industrial sectors and previous studies have reported that exposure of the lungs to ZnO-NPs induces both acute and/or chronic pulmonary inflammation, but the exact mechanism underlying such response remains elusive. This study investigated the role of nuclear factor-erythroid 2-related factor (Nrf2) in pulmonary inflammation induced by exposure to ZnO-NPs using Nrf2 null (Nrf2-/-) mice. METHODS: Twenty-four male Nrf2-/- mice and thirty male wild type C57BL/6 J mice were divided into three groups of eight and ten each respectively, and exposed once to ZnO-NPs at 0, 10, 30 µg/mouse by pharyngeal aspiration. At 14 days after the exposure to ZnO-NPs, bronchoalveolar lavage fluid (BALF) and lungs were collected to quantify protein level and the number of inflammatory cells. The mRNA levels of Nrf2-dependent antioxidant enzymes and inflammatory cytokines in lung tissue were measured. RESULTS: Exposure to ZnO-NPs dose-dependently increased the number of total cells, macrophages, lymphocytes and eosinophils in BALF both in Nrf2-/- mice and wild type mice, but the magnitude of increase was significantly higher in Nrf2-/- mice than wild type mice. The number of neutrophils in BALF increased in Nrf2-/- mice, being accompanied by marginal trend of increase in mRNA expression of MIP-2, neutrophil chemoattractant, but such changes were not observed in wild type mice. Exposure to ZnO-NPs did not dose-dependently increase mRNA level of Nrf2-dependent antioxidant enzymes both in Nrf2-/- mice and wild type mice. CONCLUSION: Pharyngeal aspiration of ZnO-NPs induced infiltration of inflammatory cells in the lung of mice, but minimally induced Nrf2-dependent antioxidant enzymes. The results suggest that Nrf2 play a role in negative regulation on ZnO-NP exposure-induced neutrophil migration, but does not demonstrate that the regulation is through suppression of oxidative stress.

Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 µM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.

Role of the ISR-ATF4 pathway and its cross talk with Nrf2 in mitochondrial quality control.

Recent investigations have clarified the importance of mitochondria in various age-related degenerative diseases, including late-onset Alzheimer's disease and Parkinson's disease. Although mitochondrial disturbances can be involved in every step of disease progression, several observations have demonstrated that a subtle mitochondrial functional disturbance is observed preceding the actual appearance of pathophysiological alterations and can be the target of early therapeutic intervention. The signals from damaged mitochondria are transferred to the nucleus, leading to the altered expression of nuclear-encoded genes, which includes mitochondrial proteins (i.e., mitochondrial retrograde signaling). Mitochondrial retrograde signaling improves mitochondrial perturbation (i.e., mitohormesis) and is considered a homeostatic stress response against intrinsic (ex. aging or pathological mutations) and extrinsic (ex. chemicals and pathogens) stimuli. There are several branches of the mitochondrial retrograde signaling, including mitochondrial unfolded protein response (UPRMT), but recent observations increasingly show the importance of the ISR-ATF4 pathway in mitochondrial retrograde signaling. Furthermore, Nrf2, a master regulator of the oxidative stress response, interacts with ATF4 and cooperatively upregulates a battery of antioxidant and antiapoptotic genes while repressing the ATF4-mediated proapoptotic gene, CHOP. In this review article, we summarized the upstream and downstream mechanisms of ATF4 activation during mitochondrial stresses and disturbances and discuss therapeutic intervention against degenerative diseases by using Nrf2 activators.

Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-κB ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced in vivo without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts in vitrovia attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of Mafb, a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-γ coactivator 1ß and mitochondrial gene expression and up-regulated Irf8 expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.-Sakai, E., Morita, M., Ohuchi, M., Kido, M. A., Fukuma, Y., Nishishita, K., Okamoto, K., Itoh, K., Yamamoto, M., Tsukuba, T. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K.

Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.

The role of NUB1 in α-synuclein degradation in Lewy body disease model mice.

Abnormal α-synuclein is deposited in neuronal cytoplasmic inclusions and presynapses in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NUB1 is accumulated in these specific regions together with abnormal α-synuclein and that NUB1 is able to inhibit α-synuclein aggregation in cultured cells. We therefore created transgenic (Tg) mice expressing both NUB1 and abnormal α-synuclein to investigate the role of NUB1 on degradation of abnormal α-synuclein in vivo. Immunohistochemical and biochemical studies confirmed that NUB1 was over-expressed in neurons of mice expressing NUB1 (NUB1 Tg), and both NUB1 and abnormal α-synuclein (double Tg). NUB1 levels were increased by 4.7-fold in NUB1 Tg mice compared with wild type mice. Unexpectedly, normal and abnormal α-synuclein levels were unchanged between abnormal α-synuclein Tg mice (Lewy body disease model mice) and double Tg mice, and pathological observations were almost similar between them. Finally, we found that the levels of insoluble α-synuclein were lower and those of some chaperone molecules were higher in double Tg mice compared with abnormal α-synuclein Tg mice. These results suggest that increased levels of NUB1 play a potential role in degradation of detergent-insoluble α-synuclein in vivo, although it is insufficient to degrade abnormal α-synuclein in Lewy body disease model mice.

Non-coding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding.

Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5' to 3') eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction.

Trehalose intake induces chaperone molecules along with autophagy in a mouse model of Lewy body disease.

The accumulation of mis-folded and/or abnormally modified proteins is a major characteristic of many neurodegenerative diseases. In Lewy body disease (LBD), which includes Parkinson's disease and dementia with Lewy bodies, insoluble α-synuclein is widely deposited in the presynaptic terminals as well as in the neuronal cytoplasm in distinct brain regions. It is well known that the autophagy-lysosome system serves as an efficient degradation pathway for abnormal molecules within cells. To test the possibility that activated autophagy can degrade abnormal molecules, we investigated the effect of trehalose on abnormal aggregation of α-synuclein in a model of LBD. Trehalose is a natural disaccharide composed of two glucose units and functions as an autophagy inducer. Consistent with previous studies, trehalose increased level of the autophagosomal protein LC3, especially a lipidated form LC3-II in cultured cells and mice brain. Also, trehalose increased levels of several chaperon molecules, such as HSP90 and SigmaR1, in the brains of LBD model mice. Further studies revealed that level of detergent-insoluble α-synuclein was suppressed in mice following oral administration of trehalose, despite an apparent alteration was not observed regarding abnormal aggregation of α-synuclein. These results suggest that the oral intake of trehalose modulates propensity of molecules prior to aggregation formation.

Role of the Keap1/Nrf2 pathway in neurodegenerative diseases.

As the elderly population increases, a growing number of individuals suffer from age-associated neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Oxidative stress is considered to play a crucial role in the pathogenesis of age-related diseases. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is activated by oxidative stress and regulates the expression of a variety of antioxidant enzymes and proteins that exert cytoprotective effects against oxidative stress. Numerous studies have addressed the role of Nrf2 in age-related diseases, including neurodegenerative diseases, using animal or in vitro cell culture models. Here, we introduce the role of oxidative stress in the pathogenesis of neurodegenerative diseases and critically examine the recent findings concerning the role for Nrf2 in the amelioration of AD and PD. Nrf2 not only regulates antioxidant proteins but also regulates the genes associated with autophagy and nerve growth factor signaling. Current research unequivocally demonstrates that the activation of the Nrf2 pathway is a promising novel strategy for the prevention and modification of neurodegenerative diseases.

Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter.

Using a luciferase reporter assay, we previously demonstrated that a Z-DNA-forming sequence of alternating thymine-guanine repeats in the human heme oxygenase-1 gene (HO-1) promoter is involved in nuclear factor erythroid-derived 2 (NF-E2)-related factor 2 (Nrf2)-mediated HO-1 promoter activation. However, the actual Z-DNA formation in this native genomic locus has not been experimentally demonstrated. To detect Z-DNA formation in vivo, we generated a construct containing the Z-DNA-binding domain of human adenosine deaminase acting on double-stranded RNA 1 fused with enhanced green fluorescence protein, designated as the Z-probe. A chromatin immunoprecipitation assay using an anti-GFP antibody showed that the Z-probe detects the well-characterized Z-DNA formation in the CSF1 promoter. Using this detection system, we demonstrated that the glutathione-depleting agent, diethyl maleate, induced Nrf2-dependent Z-DNA formation in the HO-1 promoter, but not in the thioredoxin reductase 1 gene promoter. Moreover, a time course analysis revealed that Z-DNA formation precedes HO-1 transcriptional activation. Concurrent with Z-DNA formation, nucleosome occupancy was reduced, and the recruitment of RNA polymerase II was enhanced in the HO-1 promoter region, suggesting that Z-DNA formation enhances HO-1 gene transcription. Furthermore, Nrf2-induced BRG1 recruitment to the HO-1 promoter temporarily occurred simultaneously with Z-DNA formation. Thus, these results implicate Nrf2-dependent Z-DNA formation in HO-1 transcriptional activation and suggest the involvement of BRG1 in Z-DNA formation.

Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria.

Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Carbocisteine reduces virus-induced pulmonary inflammation in mice exposed to cigarette smoke.

Carbocisteine (S-CMC) inhibits viral infection and prevents acute exacerbation of chronic obstructive pulmonary disease. We recently demonstrated the protective effects of NF-E2-related factor (Nrf) 2 against influenza virus (FluV)-induced pulmonary inflammation in mice exposed to cigarette smoke (CS). In our current study, we investigated the effects of S-CMC on Nrf2 activation in cultured macrophages, and in mice infected with influenza after exposure to CS. Nuclear translocation of Nrf2 and the expression of Nrf2-targeted antioxidant genes, such as heavy and light subunits of γ glutamyl cysteine synthetase and heme oxigenase-1, were enhanced in a dose-dependent manner after treatment with S-CMC in peritoneal and alveolar macrophages of wild-type mice, but not in those of Nrf2-deficient mice. Nuclear translocation of Nrf2 in macrophages was inhibited by the phosphatidylinositol 3-kinase inhibitor, LY294002. Phosphorylated Akt, Nrf2, and heme oxigenase-1 were induced in the alveolar macrophages of the lungs in wild-type mice after S-CMC administration. The extent of oxidative stress, inflammatory cell infiltration, pulmonary edema, and goblet cell hyperplasia was suppressed by S-CMC administration in the lungs of wild-type mice after exposure to both CS and FluV. Our findings suggest that S-CMC reduces pulmonary inflammation and mucus overproduction in mice exposed to CS after infection with FluV via the activation of Nrf2.

Transforming growth factor-ß induces transcription factors MafK and Bach1 to suppress expression of the heme oxygenase-1 gene.

Transforming growth factor-ß (TGF-ß) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-ß suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-ß did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-ß induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-ß-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-ß suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-ß is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.