JDP2 is directly regulated by ATF4 and modulates TRAIL sensitivity by suppressing the ATF4-DR5 axis.

Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.

Association of single nucleotide polymorphisms in the NRF2 promoter with vascular stiffness with aging.

PURPOSE: Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (-617C>A; hereafter called SNP-617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP-617 affects vascular stiffness with aging in apparently healthy people. METHODS: Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP-617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP-617 on other cardiovascular and blood test measurements. RESULTS: We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP-617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP-617 accelerates the incremental ratio of baPWV with age. CONCLUSIONS: This study is the first to show that SNP-617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.

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.

Increase of Tumor Infiltrating γδ T-cells in Pancreatic Ductal Adenocarcinoma Through Remodeling of the Extracellular Matrix by a Hyaluronan Synthesis Suppressor, 4-Methylumbelliferone.

OBJECTIVES: Desmoplastic changes of extracellular matrix (ECM) containing large amounts of hyaluronan (HA) are of interest in chemo- and immunoresistance of pancreatic ductal adenocarcinoma (PDAC). The goal of this study was to evaluate the effects of 4-methylumbelliferone (MU), a selective inhibitor of HA, on ECM and to examine how MU affects adoptive immunotherapy. METHODS: The effect of MU on cell proliferation, HA synthesis and formation of ECM were investigated in four PDAC cell lines. In addition, the cytotoxicity of γδ T-cell-rich peripheral blood mononuclear cells (PBMCs) collected from healthy donors and stimulated with zoledronate and interleukin-2 was examined in the presence of MU. The amount of HA and tumor-infiltrating lymphocytes were also investigated in mice xenograft models. RESULTS: In vitro, 1.0 mM MU inhibited cell proliferation by 45-70% and HA synthesis by 55-80% in all four PDAC cell lines, and enhanced γδ T-cell-rich PBMC-mediated cytotoxicity against PDAC cells. In vivo, MU reduced intratumoral HA and promoted infiltration of inoculated γδ T-cells into tumor tissue, and consequently suppressed tumor growth. CONCLUSIONS: 4-methylumbelliferone may be an effective immunosensitizer against PDAC through induction of structural changes in the ECM.

Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease.

Iron has played an important role in energy production since the beginning of life, as iron-catalyzed redox reactions are required for energy production. Oxygen, a highly efficient electron acceptor with high reduction potential, facilitates highly efficient energy production in eukaryotic cells. However, the increasing atmospheric oxygen concentration produces new threats to the organism, as oxygen reacts with iron and produces reactive oxygen species unless its levels are strictly regulated. As the size of multicellular organisms increases, these organisms must transport oxygen to the peripheral tissues and begin to employ red blood cells containing hemoglobin. This system is potentially a double-edged sword, as hemoglobin autoxidation occurs at a certain speed and releases free iron into the cytoplasm. Nrf2 belongs to the CNC transcription factor family, in which NF-E2p45 is the founding member. NF-E2p45 was first identified as a transcription factor that binds to the erythroid gene regulatory element NF-E2 located in the promoter region of the heme biosynthetic porphobilinogen deaminase gene. Human Nrf2 was also identified as a transcription factor that binds to the regulatory region of the ß-globin gene. Despite these original findings, NF-E2p45 and Nrf2 knockout mice exhibit few erythroid phenotypes. Nrf2 regulates the expression of a wide range of antioxidant and detoxification enzymes. In this review article, we describe and discuss the roles of Nrf2 in various iron-mediated bioreactions and its possible coevolution with iron and oxygen.

p62 Deficiency Enhances α-Synuclein Pathology in Mice.

In Lewy body disease (LBD) such as dementia with LBs and Parkinson's disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin-positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB-like inclusions were observed in transgenic mice that overexpressed α-synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α-synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α-synuclein aggregation.

Carnosic acid attenuates apoptosis induced by amyloid-ß 1-42 or 1-43 in SH-SY5Y human neuroblastoma cells.

Amyloid-beta (Aß) peptides, Aß 1-42 (Aß42) and Aß43 in particular, cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations. Carnosic acid (CA), a phenolic diterpene compound in the labiate herbs rosemary and sage, serves as an activator for neuroprotective and neurotrophic functions in brain cells. We investigated the effect of CA on apoptosis induced by Aß42 or Aß43 in cultured SH-SY5Y human neuroblastoma cells. Treatment of the cells with Aß42 or Aß43 (monomer, 10 µM each) induced apoptosis, which was confirmed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF). Concurrently, the Aß treatment induced the activation of caspase (Casp) cascades including an effector Casp (Casp3) and initiator Casps (Casp4, Casp8 and Casp9). Pretreatment of the cells with CA (10 µM) partially attenuated the apoptosis induced by Aß42 or Aß43. CA pretreatment also reduced the cellular oligomers of Aß42 and Aß43. These results suggest that CA suppressed the activation of Casp cascades by reducing the intracellular oligomerization of exogenous Aß42/43 monomer. The ingestion of an adequate amount of CA may have a potential in the prevention of Aß-mediated diseases, particularly AD.

Nrf2- and ATF4-dependent upregulation of xCT modulates the sensitivity of T24 bladder carcinoma cells to proteasome inhibition.

The ubiquitin-proteasome pathway degrades ubiquitinated proteins to remove damaged or misfolded protein and thus plays an important role in the maintenance of many important cellular processes. Because the pathway is also crucial for tumor cell growth and survival, proteasome inhibition by specific inhibitors exhibits potent antitumor effects in many cancer cells. xCT, a subunit of the cystine antiporter system xc (-), plays an important role in cellular cysteine and glutathione homeostasis. Several recent reports have revealed that xCT is involved in cancer cell survival; however, it was unknown whether xCT affects the cytotoxic effects of proteasome inhibitors. In this study, we found that two stress-inducible transcription factors, Nrf2 and ATF4, were upregulated by proteasome inhibition and cooperatively enhance human xCT gene expression upon proteasome inhibition. In addition, we demonstrated that the knockdown of xCT by small interfering RNA (siRNA) or pharmacological inhibition of xCT by sulfasalazine (SASP) or (S)-4-carboxyphenylglycine (CPG) significantly increased the sensitivity of T24 cells to proteasome inhibition. These results suggest that the simultaneous inhibition of both the proteasome and xCT could have therapeutic benefits in the treatment of bladder tumors.

Phosphorylation of serine 349 of p62 in Alzheimer's disease brain.

BACKGROUND: Extensive research on p62 has established its role in oxidative stress, protein degradation and in several diseases such as Paget's disease of the bone, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Importantly, previous studies showed that p62 binds directly to Keap1, which is a ubiquitin E3 ligase responsible for degrading Nrf2. Indeed, colocalisation of p62 and Keap1 occurs in tumorigenesis and neurodegeneration. A serine (S) residue in the Keap1-interacting region of p62 is phosphorylated in hepatocellular carcinoma, and this phosphorylation contributes to tumour growth through the higher affinity of p62 to Keap1. However, it remains largely unknown whether p62 is phosphorylated in the Keap1-interacting region under neurodegenerative conditions. RESULTS: To answer this question, we generated an antibody against phosphorylated S349 (P-S349) of p62 and showed that S349 is phosphorylated following disruption of protein degradation. In particular, the ratio of P-S349 to total p62 levels was significantly increased in the brains with Alzheimer's disease (AD) compared with controls. We also compared the reactivity of the P-S349 antibody with P-S403 of p62 and showed that these two phosphorylated sites on p62 cause different responses with proteasome inhibition and show distinct localisation patterns in AD brains. In addition to disruption of protein degradation systems, activation of oxidative stress can induce P-S349. CONCLUSION: These results support the hypothesis that disruption of protein degradation systems and sustained activation of the Keap1-Nrf2 system occur in the brains with AD.

Carnosic acid suppresses the production of amyloid-ß 1-42 and 1-43 by inducing an α-secretase TACE/ADAM17 in U373MG human astrocytoma cells.

Amyloid beta (Aß) peptides are key molecules in the pathogenesis of Alzheimer's disease (AD). The sequential cleavage of amyloid precursor protein (APP) by the ß- and γ-secretases generates Aß peptides; however, the alternate cleavage of APP by the α- and γ-secretases decreases Aß production. We previously reported that carnosic acid (CA), a phenolic diterpene compound found in the labiate herbs rosemary and sage, suppresses Aß (1-40 and 1-42) production by activating α-secretase in cultured SH-SY5Y human neuroblastoma cells (Neurosci. Res. 2013; 75: 94-102). Here, we investigated the effect of CA on the production of Aß peptides (1-40, 1-42 and 1-43) in U373MG human astrocytoma cells. The treatment of cells with CA suppressed Aß40/42/43 release (55-71% decrease at 50µM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17); however, the ß-secretase BACE1 (ß-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα release enhanced by CA and partially recovered the CA-suppressed Aß40/42/43 release. These results suggest that CA reduces Aß production, at least partially, by activating TACE in human astroglial cells. The use of CA may have a potential in the prevention of Aß-mediated 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.

Carnosic acid suppresses the production of amyloid-ß 1-42 by inducing the metalloprotease gene TACE/ADAM17 in SH-SY5Y human neuroblastoma cells.

A hallmark of Alzheimer's disease (AD) is the aggressive appearance of plaques of amyloid beta (Aß) peptides, which result from the sequential cleavage of amyloid precursor protein (APP) by the ß- and γ-secretases. Aß production is evaded by alternate cleavage of APP by the α- and γ-secretases. Carnosic acid (CA) has been proven to activate the transcription factor Nrf2, a main regulator of the antioxidant response. We investigated the effects of CA on the production of Aß 1-42 peptide (Aß42) and on the expressions of the related genes in SH-SY5Y human neuroblastoma cells. The treatment of cells with CA suppressed Aß42 secretion (61% suppression at 30µM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17) significantly and another α-secretase ADAM10 marginally; however, the ß-secretase BACE1 (ß-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα secretion enhanced by CA and partially recovered the CA-suppressed Aß42 secretion. These results suggest that CA reduces Aß42 production by activating TACE without promoting BACE1 in human neuroblastoma cells. The use of CA may have a potential in the prevention of Aß-mediated diseases, particularly AD.

Nrf2 in bone marrow-derived cells positively contributes to the advanced stage of atherosclerotic plaque formation.

Atherosclerosis is the major etiology underlying myocardial infarction and stroke, and strategies for preventing atherosclerosis are urgently needed. In the context of atherosclerosis, the deletion of the Nrf2 gene, which encodes a master regulator of the oxidative stress response in mammals, reportedly attenuates atherosclerosis formation. However, the precise mechanisms of protection against atherosclerosis are largely unknown. To further clarify the role of Nrf2 in atherosclerosis in vivo, we performed a time course analysis of atherosclerosis development utilizing an ApoE knockout (KO) mouse model. The results demonstrate that oil red O-stainable lesions were similar in size 5 weeks after the initiation of an HFC (high fat and high cholesterol) diet, but the lesions were markedly attenuated in the Nrf2 and ApoE double KO mice (A0N0 mice) compared with the lesions in the ApoE KO mice (A0N2 mice) at 12 weeks. Consistent with these results, the immunohistochemical analysis revealed that Nrf2 activation is observed in late-stage atherosclerotic plaques but not in earlier lesions. The RT-qPCR analysis of 12-week atherosclerotic plaques revealed that Nrf2 target genes, such as Ho-1 and SLPI, are expressed at significantly lower levels in the A0N0 mice compared with the A0N2 mice, and this change was associated with a decreased expression of macrophage M1-subtype genes Arginase II and inducible NO synthase in the A0N0 mice. Furthermore, the bone marrow (BM) transplantation (BMT) analysis revealed that the Nrf2 activity in the BM-derived cells contributed to lesion formation. Therefore, our study has characterized the positive role of Nrf2 in the BM-derived cells during the development of atherosclerosis, which suggests that Nrf2 may influence the inflammatory reactions in the plaques.

Methylation of the KEAP1 gene promoter region in human colorectal cancer.

BACKGROUND: The Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated. METHODS: We used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1. RESULTS: DNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs. CONCLUSION: Hypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

Nrf2 regulates NGF mRNA induction by carnosic acid in T98G glioblastoma cells and normal human astrocytes.

Nerve growth factor (NGF) is a neurotrophic factor that plays an important role in neuronal cell development and survival. Carnosic acid (CA), a hydrophobic constituent of the herb rosemary, induces NGF production in human T98G glioblastoma cells, but the mechanism through which it works remains unknown. In the present study, we found a redox-sensitive transcription factor, Nrf2, which coordinates the expression of cytoprotective phase 2 genes, also participates in CA-inducible NGF expression. In T98G cells, CA caused NGF gene induction in a dose- and time-dependent manner without altering NGF mRNA stability. Simultaneously, CA increased Nrf2 nuclear accumulation and activated expression of prototypical Nrf2 target genes such as haem oxygenase 1 (HO-1) and thioredoxin reductase 1 (TXNRD1). Knockdown of endogenous Nrf2 by Nrf2-specific siRNA significantly reduced constitutive and CA-inducible NGF gene expression. In addition, NGF gene expression was enhanced by knockdown of Keap1, an Nrf2 inhibitor, in the absence of CA. Furthermore, CA induced NGF expression in normal human astrocytes in an Nrf2-dependent manner. These results highlight a role of Nrf2 in NGF gene expression in astroglial cells.

Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages.

Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation.

Suppression of AhR signaling pathway is associated with the down-regulation of UDP-glucuronosyltransferases during BBN-induced urinary bladder carcinogenesis in mice.

Down-regulation of carcinogen detoxifying enzymes might be a critical factor in tumour formation by increasing the carcinogen concentration in the target organ. Previous reports revealed that the expression of UGT1A mRNA is either lost or decreased in certain human cancer tissues, including urinary bladder cancer. To elucidate this down-regulation mechanism, we used an N-nitrosobutyl (4-hydroxybutyl) amine (BBN)-induced mouse urinary bladder carcinogenesis model. Similar to human cancer, the expressions of Ugt1a6, Ugt1a9 and total Ugt1a mRNA in the BBN-induced bladder cancer were markedly decreased compared with those of normal mice. BBN down-regulated the basal Ugt1a mRNA expression in a time-dependent manner and this was reversible in the first 2 weeks of BBN treatment. However, after 4 weeks of BBN treatment the repression became persistent after the cessation of BBN treatment. Aryl hydrocarbon receptor (AhR) regulates the constitutive and inducible expression of Ugt1a mRNA. We found that the constitutive Ugt1a mRNA expression is decreased in the bladder of AhR knockout (KO) mice. Furthermore, BBN-induced Ugt1a down-regulation was lost in AhR KO mice, and the canonical AhR target gene Cyp1a1 was similarly down-regulated by BBN in the bladder. These results demonstrate that BBN repressed Ugt1a mRNA expression via suppression of AhR signaling pathway during BBN-induced carcinogenesis.

Role of Nrf2 and p62/ZIP in the neurite outgrowth by carnosic acid in PC12h cells.

Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can permeate the blood-brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer's disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA-induced p62/ZIP expression in an Nrf2-dependent manner, while the CA-induced neural differentiation was suppressed by p62/ZIP knockdown. Furthermore, CA-induced ARE activation was attenuated both by p62/ZIP knockdown and a Trk signal inhibitor. These results suggest that the CA induction of p62/ZIP by Nrf2 enhances TrkA signaling which subsequently potentiates Nrf2 pathway. This is the first demonstration that activation of the Nrf2-p62/ZIP pathway by a low-molecular natural electrophilic compound plays important roles in TrkA-mediated neural differentiation and may represent the common molecular mechanism for neurotrophic activities of electrophilic compounds.

Hypersensitivity of aryl hydrocarbon receptor-deficient mice to lipopolysaccharide-induced septic shock.

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is known to mediate a wide variety of pharmacological and toxicological effects caused by polycyclic aromatic hydrocarbons. Recent studies have revealed that AhR is involved in the normal development and homeostasis of many organs. Here, we demonstrate that AhR knockout (AhR KO) mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, mainly due to the dysfunction of their macrophages. In response to LPS, bone marrow-derived macrophages (BMDM) of AhR KO mice secreted an enhanced amount of interleukin-1beta (IL-1beta). Since the enhanced IL-1beta secretion was suppressed by supplementing Plasminogen activator inhibitor-2 (Pai-2) expression through transduction with Pai-2-expressing adenoviruses, reduced Pai-2 expression could be a cause of the increased IL-1beta secretion by AhR KO mouse BMDM. Analysis of gene expression revealed that AhR directly regulates the expression of Pai-2 through a mechanism involving NF-kappaB but not AhR nuclear translocator (Arnt), in an LPS-dependent manner. Together with the result that administration of the AhR ligand 3-methylcholanthrene partially protected mice with wild-type AhR from endotoxin-induced death, these results raise the possibility that an appropriate AhR ligand may be useful for treating patients with inflammatory disorders.