Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor n... more Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which upregulates the expression of genes important for detoxification, glutathione synthesis, and defense against oxidative damage. Stress-activated MAP kinases p38, ERK, and JNK cooperate in the efficient nuclear accumulation of Nrf2 in a cell-type-dependent manner. Activation of p38 induces membrane trafficking of a glutathione sensor neutral sphingomyelinase 2, which generates ceramide upon depletion of cellular glutathione. We previously proposed that caveolin-1 in lipid rafts provides a signaling hub for the phosphorylation of Nrf2 by ceramide-activated PKCζ and casein kinase 2 to stabilize Nrf2 and mask a nuclear export signal. We further propose a mechanism of facilitated Nrf2 nuclear translocation by ERK and JNK. ERK and JNK phosphorylation of Nrf2 induces the association of prolyl cis/trans isomerase Pin1, which specifically recognizes ...
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset ene... more control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset energy metabolism in astrocytes via brain-derived neurotrophic factor.
Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwi... more Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwide. However, details of pathogenetic mechanisms remain unknown. Deletion of both p62/Sqstm1 and Nrf2 genes spontaneously led to the development of NASH in mice fed a normal chow and was associated with liver tumorigenesis. The pathogenetic mechanism (s) underlying the NASH development was investigated in p62:Nrf2 double-knockout (DKO) mice. DKO mice showed massive hepatomegaly and steatohepatitis with fat accumulation and had hyperphagia-induced obesity coupled with insulin resistance and adipokine imbalance. They also showed dysbiosis associated with an increased proportion of gram-negative bacteria species and an increased lipopolysaccharide (LPS) level in feces. Intestinal permeability was elevated in association with both epithelial damage and decreased expression levels of tight junction protein zona occludens-1, and thereby LPS levels were increased in serum. For Kupffer cells, the...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a sele... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons in the brain and spinal cord. Multiple toxicity pathways, such as oxidative stress, misfolded protein accumulation, and dysfunctional autophagy, are implicated in the pathogenesis of ALS. However, the molecular basis of the interplay between such multiple factors in vivo remains unclear. Here, we report that two independent ALS-linked autophagy-associated gene products; SQSTM1/p62 and ALS2/alsin, but not antioxidant-related factor; NFE2L2/Nrf2, are implicated in the pathogenesis in mutant SOD1 transgenic ALS models. We generated SOD1(H46R) mice either on a Nfe2l2-null, Sqstm1-null, or Sqstm1/Als2-double null background. Loss of SQSTM1 but not NFE2L2 exacerbated disease symptoms. A simultaneous inactivation of SQSTM1 and ALS2 further accelerated the onset of disease. Biochemical analyses revealed that loss of SQSTM1 increased the level of insoluble SOD1 at the ...
Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms... more Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system [Formula: see text], which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the [Formula: see text] transport system leading to increases in cellular GS...
The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cos... more The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative nontoxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.
The anticancer agent cis-diamminedichloroplatinum (cisplatin) is a first-line chemotherapeutic ag... more The anticancer agent cis-diamminedichloroplatinum (cisplatin) is a first-line chemotherapeutic agent for oral cancer. Cell exposure to cisplatin is associated with increased oxidative stress and post-translational changes in components of apoptosis pathways, including p38 Mitogen-activated protein kinase (MAPK), c-Jun-NH2-kinase (JNK), and extracellular signal-regulated kinase (ERK). Peroxiredoxin (Prx) I is an oxidative stress-inducible protein expressed in many tissues and important for reducing reactive oxygen species in vivo; however, whether Prx I helps protect cells from cisplatin injury is unknown. In this report, we examined the effects of Prx I on cell sensitivity to cisplatin-induced apoptosis. Mouse embryo fibroblasts (MEFs) derived from Prx I-deficient mice showed increased cisplatin-induced apoptosis compared with wild-type MEFs. Cisplatin treatment also led to increased activation of p38 MAPK and JNK, and reduced ERK phosphorylation in Prx I-deficient MEFs compared with wild-type MEFs. Furthermore, JNK-and ERK-specific inhibitors protected the Prx I-deficient MEFs from cisplatin-induced apoptosis, but Prx I-deficient MEFs remained more sensitive than wild-type MEFs when treated with a p38 MAPK-specific inhibitor. These findings indicate that Prx I modulates the cisplatin-evoked activation of MAPKs that lead to apoptosis, and Prx I may thus represent a useful target as a protective therapy against cisplatin cytotoxicity.
Activated macrophages express high levels of Nrf2, a transcription factor that positively regulat... more Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), it was observed that macrophages from pleural lavage accumulate 15d-PGJ 2 . We show that in mouse peritoneal macrophages 15d-PGJ 2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrop...
The anti-inflammatory properties of transforming growth factor-1 (TGF-1) account for its protec... more The anti-inflammatory properties of transforming growth factor-1 (TGF-1) account for its protection against atherosclerotic plaque rupture. This study investigates whether activation of the Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) transcription pathway is involved in TGF-1 mediated induction of the antioxidant enzyme heme oxygenase-1 (HO-1) in smooth muscle cells (SMC). Human aortic smooth muscle cells (HAoSMC) or wild-type and Nrf2-deficient mouse (MAoSMC) aortic SMC were treated with TGF-1 (2.5-10 ng/ml, 0-24 hrs). We report the first evidence that TGF-1 induces Nrf2 mediated HO-1 expression and antioxidant response element activity, which was paralleled by enhanced superoxide production and expression of the NAD(P)H oxidase subunit p22 phox. TGF-1 failed to induce HO-1 expression in MAoSMC derived from Nrf2-deficient mice, and HO-1 induction by TGF-1 in HAoSMC was attenuated by inhibition of extracellular signal regulated kinase or c-jun-N-terminal kinase but not p38 mitogen activated protein kinase. Inhibition of NAD(P)H oxidase or scavenging of superoxide diminished HO-1 induction in response to TGF-1. The oxidative stress agents glucose oxidase (GOx) and diethylmaleate enhanced TGF-1 generation and HO-1 expression in HAoSMC, while antagonism of TGF-1 signalling by adenoviral Smad7 overexpression attenuated their induction of HO-1. Pre-treatment of HAoSMC with TGF-1 reduced nuclear translocation of the pro-apoptotic mediator p53 elicited by GOx. Our findings demonstrate that Nrf2 is a new target of TGF-1 signalling in the vasculature which may contribute to the atheroprotective properties attributed to this growth factor.
Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers wit... more Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O 2 in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by ϳ70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na ؉-independent anionic amino acid transporter named system x c Ϫ. System x c Ϫ is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x c Ϫ activity. A putative NF-B binding site was found in the 5-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-B binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not ␥-glutamylcysteine synthetase. These results suggest the importance of system x c Ϫ in antioxidant defense in macrophages exposed to LPS and oxidative stress.
Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseas... more Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione Stransferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophileand oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system x c Ϫ) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system x c Ϫ activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wideranging metabolic response to oxidative stress.
Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins... more Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Cells have developed a network of pathways aimed at maintaining proteostasis. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins, which occur in response to LPS stimulation or puromycin treatment. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that oc...
Rodents have brownish-yellow incisors whose color represents their iron content. Iron is deposite... more Rodents have brownish-yellow incisors whose color represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region-leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2-deficient mice show decolorization of the incisors. While incisors of wild type mice were brownish yellow, incisors of Nrf2-deficient mice were grayish white in color. Micro X-ray imaging analysis revealed that the iron content in Nrf2-deficient mouse incisors were significantly decreased compared to that of wild type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2-deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2-null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2-deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition onto the enamel surface.
Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role ... more Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity.
CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (ox... more CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx...
Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and... more Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin-and LC3-binding protein ''p62'' regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific. (D) The level of Nrf2 in liver nuclear fraction. Nuclear fractions were prepared from liver of the indicated genotypes at 12 days post-pIpC injection, subjected to SDS-PAGE, and analyzed by immunoblotting with antibodies against Nrf2 and Lamin B (as a control). Data represent two independent mice.
Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor n... more Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which upregulates the expression of genes important for detoxification, glutathione synthesis, and defense against oxidative damage. Stress-activated MAP kinases p38, ERK, and JNK cooperate in the efficient nuclear accumulation of Nrf2 in a cell-type-dependent manner. Activation of p38 induces membrane trafficking of a glutathione sensor neutral sphingomyelinase 2, which generates ceramide upon depletion of cellular glutathione. We previously proposed that caveolin-1 in lipid rafts provides a signaling hub for the phosphorylation of Nrf2 by ceramide-activated PKCζ and casein kinase 2 to stabilize Nrf2 and mask a nuclear export signal. We further propose a mechanism of facilitated Nrf2 nuclear translocation by ERK and JNK. ERK and JNK phosphorylation of Nrf2 induces the association of prolyl cis/trans isomerase Pin1, which specifically recognizes ...
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset ene... more control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset energy metabolism in astrocytes via brain-derived neurotrophic factor.
Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwi... more Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwide. However, details of pathogenetic mechanisms remain unknown. Deletion of both p62/Sqstm1 and Nrf2 genes spontaneously led to the development of NASH in mice fed a normal chow and was associated with liver tumorigenesis. The pathogenetic mechanism (s) underlying the NASH development was investigated in p62:Nrf2 double-knockout (DKO) mice. DKO mice showed massive hepatomegaly and steatohepatitis with fat accumulation and had hyperphagia-induced obesity coupled with insulin resistance and adipokine imbalance. They also showed dysbiosis associated with an increased proportion of gram-negative bacteria species and an increased lipopolysaccharide (LPS) level in feces. Intestinal permeability was elevated in association with both epithelial damage and decreased expression levels of tight junction protein zona occludens-1, and thereby LPS levels were increased in serum. For Kupffer cells, the...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a sele... more Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons in the brain and spinal cord. Multiple toxicity pathways, such as oxidative stress, misfolded protein accumulation, and dysfunctional autophagy, are implicated in the pathogenesis of ALS. However, the molecular basis of the interplay between such multiple factors in vivo remains unclear. Here, we report that two independent ALS-linked autophagy-associated gene products; SQSTM1/p62 and ALS2/alsin, but not antioxidant-related factor; NFE2L2/Nrf2, are implicated in the pathogenesis in mutant SOD1 transgenic ALS models. We generated SOD1(H46R) mice either on a Nfe2l2-null, Sqstm1-null, or Sqstm1/Als2-double null background. Loss of SQSTM1 but not NFE2L2 exacerbated disease symptoms. A simultaneous inactivation of SQSTM1 and ALS2 further accelerated the onset of disease. Biochemical analyses revealed that loss of SQSTM1 increased the level of insoluble SOD1 at the ...
Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms... more Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system [Formula: see text], which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the [Formula: see text] transport system leading to increases in cellular GS...
The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cos... more The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative nontoxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.
The anticancer agent cis-diamminedichloroplatinum (cisplatin) is a first-line chemotherapeutic ag... more The anticancer agent cis-diamminedichloroplatinum (cisplatin) is a first-line chemotherapeutic agent for oral cancer. Cell exposure to cisplatin is associated with increased oxidative stress and post-translational changes in components of apoptosis pathways, including p38 Mitogen-activated protein kinase (MAPK), c-Jun-NH2-kinase (JNK), and extracellular signal-regulated kinase (ERK). Peroxiredoxin (Prx) I is an oxidative stress-inducible protein expressed in many tissues and important for reducing reactive oxygen species in vivo; however, whether Prx I helps protect cells from cisplatin injury is unknown. In this report, we examined the effects of Prx I on cell sensitivity to cisplatin-induced apoptosis. Mouse embryo fibroblasts (MEFs) derived from Prx I-deficient mice showed increased cisplatin-induced apoptosis compared with wild-type MEFs. Cisplatin treatment also led to increased activation of p38 MAPK and JNK, and reduced ERK phosphorylation in Prx I-deficient MEFs compared with wild-type MEFs. Furthermore, JNK-and ERK-specific inhibitors protected the Prx I-deficient MEFs from cisplatin-induced apoptosis, but Prx I-deficient MEFs remained more sensitive than wild-type MEFs when treated with a p38 MAPK-specific inhibitor. These findings indicate that Prx I modulates the cisplatin-evoked activation of MAPKs that lead to apoptosis, and Prx I may thus represent a useful target as a protective therapy against cisplatin cytotoxicity.
Activated macrophages express high levels of Nrf2, a transcription factor that positively regulat... more Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), it was observed that macrophages from pleural lavage accumulate 15d-PGJ 2 . We show that in mouse peritoneal macrophages 15d-PGJ 2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrop...
The anti-inflammatory properties of transforming growth factor-1 (TGF-1) account for its protec... more The anti-inflammatory properties of transforming growth factor-1 (TGF-1) account for its protection against atherosclerotic plaque rupture. This study investigates whether activation of the Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) transcription pathway is involved in TGF-1 mediated induction of the antioxidant enzyme heme oxygenase-1 (HO-1) in smooth muscle cells (SMC). Human aortic smooth muscle cells (HAoSMC) or wild-type and Nrf2-deficient mouse (MAoSMC) aortic SMC were treated with TGF-1 (2.5-10 ng/ml, 0-24 hrs). We report the first evidence that TGF-1 induces Nrf2 mediated HO-1 expression and antioxidant response element activity, which was paralleled by enhanced superoxide production and expression of the NAD(P)H oxidase subunit p22 phox. TGF-1 failed to induce HO-1 expression in MAoSMC derived from Nrf2-deficient mice, and HO-1 induction by TGF-1 in HAoSMC was attenuated by inhibition of extracellular signal regulated kinase or c-jun-N-terminal kinase but not p38 mitogen activated protein kinase. Inhibition of NAD(P)H oxidase or scavenging of superoxide diminished HO-1 induction in response to TGF-1. The oxidative stress agents glucose oxidase (GOx) and diethylmaleate enhanced TGF-1 generation and HO-1 expression in HAoSMC, while antagonism of TGF-1 signalling by adenoviral Smad7 overexpression attenuated their induction of HO-1. Pre-treatment of HAoSMC with TGF-1 reduced nuclear translocation of the pro-apoptotic mediator p53 elicited by GOx. Our findings demonstrate that Nrf2 is a new target of TGF-1 signalling in the vasculature which may contribute to the atheroprotective properties attributed to this growth factor.
Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers wit... more Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O 2 in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by ϳ70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na ؉-independent anionic amino acid transporter named system x c Ϫ. System x c Ϫ is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x c Ϫ activity. A putative NF-B binding site was found in the 5-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-B binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not ␥-glutamylcysteine synthetase. These results suggest the importance of system x c Ϫ in antioxidant defense in macrophages exposed to LPS and oxidative stress.
Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseas... more Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione Stransferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophileand oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system x c Ϫ) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system x c Ϫ activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wideranging metabolic response to oxidative stress.
Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins... more Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Cells have developed a network of pathways aimed at maintaining proteostasis. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins, which occur in response to LPS stimulation or puromycin treatment. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that oc...
Rodents have brownish-yellow incisors whose color represents their iron content. Iron is deposite... more Rodents have brownish-yellow incisors whose color represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region-leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2-deficient mice show decolorization of the incisors. While incisors of wild type mice were brownish yellow, incisors of Nrf2-deficient mice were grayish white in color. Micro X-ray imaging analysis revealed that the iron content in Nrf2-deficient mouse incisors were significantly decreased compared to that of wild type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2-deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2-null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2-deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition onto the enamel surface.
Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role ... more Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity.
CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (ox... more CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx...
Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and... more Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin-and LC3-binding protein ''p62'' regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific. (D) The level of Nrf2 in liver nuclear fraction. Nuclear fractions were prepared from liver of the indicated genotypes at 12 days post-pIpC injection, subjected to SDS-PAGE, and analyzed by immunoblotting with antibodies against Nrf2 and Lamin B (as a control). Data represent two independent mice.
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Papers by Tetsuro Ishii