Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Methylmercury (MeHg) is a well-known environmental neurotoxicant that causes severe brain disorders such as Minamata disease. Although some patients with Minamata disease develop olfactory dysfunction, the underlying pathomechanism is largely unknown. We examined the effects of MeHg on the olfactory system using a model of MeHg poisoning in which mice were administered 30 ppm MeHg in drinking water for 8 weeks. Mice exposed to MeHg displayed significant mercury accumulation in the olfactory pathway, including the nasal mucosa, olfactory bulb, and olfactory cortex. The olfactory epithelium was partially atrophied, and olfactory sensory neurons were diminished. The olfactory bulb exhibited an increase in apoptotic cells, hypertrophic astrocytes, and amoeboid microglia, mainly in the granular cell layer. Neuronal cell death was observed in the olfactory cortex, particularly in the ventral tenia tecta. Neuronal cell death was also remarkable in higher-order areas such as the orbitofrontal cortex. Correlation analysis showed that neuronal loss in the olfactory cortex was strongly correlated with the plasma mercury concentration. Our results indicate that MeHg is an olfactory toxicant that damages the central regions involved in odor perception. The model described herein is useful for analyzing the mechanisms and treatments of olfactory dysfunction in MeHg-intoxicated patients.

DOI： 10.1007/s00204-024-03682-w

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Other Link： https://link.springer.com/article/10.1007/s00204-024-03682-w/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The link between changes in astrocyte function and the pathological progression of Alzheimer's disease (AD) has attracted considerable attention. Interestingly, activated astrocytes in AD show abnormalities in their lipid content and metabolism. In particular, the expression of apolipoprotein E (ApoE), a lipid transporter, is decreased. Because ApoE has anti-inflammatory and amyloid β (Aβ)-metabolizing effects, the nuclear receptors, retinoid X receptor (RXR) and LXR, which are involved in ApoE expression, are considered promising therapeutic targets for AD. However, the therapeutic effects of agents targeting these receptors are limited or vary considerably among groups, indicating the involvement of an unknown pathological factor that modifies astrocyte and ApoE function. Here, we focused on the signaling lipid, sphingosine-1-phosphate (S1P), which is mainly produced by sphingosine kinase 2 (SphK2) in the brain. Using astrocyte models, we found that upregulation of SphK2/S1P signaling suppressed ApoE induction by both RXR and LXR agonists. We also found that SphK2 activation reduced RXR binding to the APOE promoter region in the nucleus, suggesting the nuclear function of SphK2/S1P. Intriguingly, suppression of SphK2 activity by RNA knockdown or specific inhibitors upregulated lipidated ApoE induction. Furthermore, the induced ApoE facilitates Aβ uptake in astrocytes. Together with our previous findings that SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons, these results indicate that SphK2/S1P signaling is a promising multifunctional therapeutic target for AD that can modulate astrocyte function by stabilizing the effects of RXR and LXR agonists, and simultaneously regulate neuronal pathogenesis.

DOI： 10.1016/j.jlr.2024.100510

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Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed epidermal growth factor-induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

DOI： 10.1016/j.jbc.2024.105679

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Toxicology  

Apoptosis is one of the hallmarks of MeHg-induced neuronal cell death; however, its molecular mechanism remains unclear. We previously reported that MeHg exposure induces neuron-specific ER stress in the mouse brain. Excessive ER stress contributes to apoptosis, and CHOP induction is considered to be one of the major mechanisms. CHOP is also increased by MeHg exposure in the mouse brain, suggesting that it correlates with increased apoptosis. In this study, to clarify whether CHOP mediates MeHg-induced apoptosis, we examined the effect of CHOP deletion on MeHg exposure in CHOP-knockout mice. Our data showed that CHOP deletion had no effect on MeHg exposure-induced weight loss or hindlimb impairment in mice, nor did it increase apoptosis or inhibit neuronal cell loss. Hence, CHOP plays little role in MeHg toxicity, and other apoptotic pathways coupled with ER stress may be involved in MeHg-induced cell death.

DOI： 10.2131/jts.49.55

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Toxicology  

Nitric oxide (NO) plays a physiological role in signal transduction and excess or chronic NO has toxic effects as an inflammatory mediator. NO reversibly forms protein S-nitrosylation and exerts toxicological functions related to disease progression. DNA methyltransferases, epigenome-related enzymes, are inhibited in enzymatic activity by S-nitrosylation. Therefore, excess or chronic NO exposure may cause disease by altering gene expression. However, the effects of chronic NO exposure on transcriptome are poorly understood. Here, we performed transcriptome analysis of A549, AGS, HEK293T, and SW48 cells exposed to NO (100 μM) for 48 hr. We showed that the differentially expressed genes were cell-specific. Gene ontology analysis showed that the functional signature of differentially expressed genes related to cell adhesion or migration was upregulated in several cell lines. Gene set enrichment analysis indicated that NO stimulated inflammation-related gene expression in various cell lines. This finding supports previous studies showing that NO is closely involved in inflammatory diseases. Overall, this study elucidates the pathogenesis of NO-associated inflammatory diseases by focusing on changes in gene expression.

DOI： 10.2131/jts.49.281

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Upregulation of nitric oxide (NO) production contributes to the pathogenesis of numerous diseases via S-nitrosylation, a post-translational modification of proteins. This process occurs due to the oxidative reaction between NO and a cysteine thiol group; however, the extent of this reaction remains unknown. S-Nitrosylation of PRMT1, a major asymmetric arginine methyltransferase of histones and numerous RNA metabolic proteins, was induced by NO donor treatment. We found that nitrosative stress leads to S-nitrosylation of cysteine 119, located near the active site, and attenuates the enzymatic activity of PRMT1. Interestingly, RNA sequencing analysis revealed similarities in the changes in expression elicited by NO and PRMT1 inhibitors or knockdown. A comprehensive search for PRMT1 substrates using the proximity-dependent biotin identification method highlighted many known and new substrates, including RNA-metabolizing enzymes. To validate this result, we selected the RNA helicase DDX3 and demonstrated that arginine methylation of DDX3 is induced by PRMT1 and attenuated by NO treatment. Our results suggest the existence of a novel regulatory system associated with transcription and RNA metabolism via protein S-nitrosylation.

DOI： 10.1016/j.jphs.2023.12.012

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer’s disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-β (Aβ), which accumulates in the patient brain. The “amyloid hypothesis” based therapy that places the aggregation and toxicity of Aβ at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. Aβ is produced from a type-I transmembrane protein, Aβ precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called βCTF), is a direct precursor of Aβ and accumulates in the AD patient’s brain to demonstrate toxicity independent of Aβ. Conventional drug discovery strategies have focused on Aβ toxicity on the “outside” of the neuron, but C99 accumulation might explain the toxicity on the “inside” of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and Aβ is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis.

DOI： 10.3390/cells12030454

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures specific areas of the brain. MeHg is known to induce oxidative and endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway has a dual nature in that it regulates and protects cells from an overload of improperly folded proteins in the ER, whereas excessively stressed cells are eliminated by apoptosis. Oxidative stress/ER stress induced by methylmercury exposure may tilt the UPR toward apoptosis, but there is little in vivo evidence of a direct link to actual neuronal cell death. Here, by using the ER stress-activated indicator (ERAI) system, we investigated the time course signaling alterations of UPR in vivo in the most affected areas, the somatosensory cortex and striatum. In the ERAI-Venus transgenic mice exposed to MeHg (30 or 50 ppm in drinking water), the ERAI signal, which indicates the activation of the cytoprotective pathway of the UPR, was only transiently enhanced, whereas the apoptotic pathway of the UPR was persistently enhanced. Furthermore, detailed analysis following the time course showed that MeHg-induced apoptosis is strongly associated with alterations in UPR signaling. Our results suggest that UPR modulation could be a therapeutic target for treating neuropathy.

DOI： 10.3390/ijms232315412

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Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'| disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported that βCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between βCTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and AppNL-G-F/NL-G-F model mice. Furthermore, the T-RAP peptide derived from the βCTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD.

DOI： 10.1016/j.isci.2022.103869

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg.

DOI： 10.1007/s00204-021-02982-9

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The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase. Several EGFR mutations and modifications have been shown to lead to abnormal self-activation, which plays a critical role in carcinogenesis. Environmental air pollutants, which are associated with cancer and respiratory diseases, can also activate EGFR. Specifically, the environmental electrophile 1,2- naphthoquinone (1,2-NQ), a component of diesel exhaust particles and particulate matter more generally, has previously been shown to impact EGFR signaling. However, the detailed mechanism of 1,2-NQ function is unknown. Here, we demonstrate that 1,2-NQ is a novel chemical activator of EGFR but not other EGFR family proteins. We found that 1,2-NQ forms a covalent bond, in a reaction referred to as N-arylation, with Lys80, which is in the ligand-binding domain. This modification activates the EGFR-Akt signaling pathway, which inhibits serum deprivation-induced cell death in a human lung adenocarcinoma cell line. Our study reveals a novel mode of EGFR pathway activation and suggests a link between abnormal EGFR activation and environmental pollutant- associated diseases such as cancer.

DOI： 10.1016/j.jbc.2021.100524

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Language：Japanese   Publisher：Japanese Pharmacological Society  

[Background] The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase whose mutation or modification leads to abnormal self-activation, which plays a critical role in carcinogenesis. Although environmental air pollutants, which are associated with cancer and respiratory diseases, can activate EGFR, the detailed mechanisms remain unknown. In this study, we addressed to clarify the mechanisms by which the air pollutant 1,2-naphthoquinone (1,2-NQ) disrupts EGFR-Akt signaling and promotes oncogenic effects.

[Methods] Phosphorylated forms of Akt, EGFR, IGF-IR, and Insulin receptor were detected by western blotting using specific antibodies. The modification sites in EGFR were identified by LC-MS/MS analysis. Cell viability was assessed by WST-8 assay.

[Results] We found that 1,2-NQ stimulates the phosphorylation of EGFR, but not IGF-IR and Insulin receptor in A549 cells. LC-MS/MS analysis revealed that 1,2-NQ forms chemical modification with EGFR at ligand-binding domain I, Lys80. In addition, phosphorylation of Akt was observed by treatment with 1,2-NQ in a concentration-dependent manner. Activation of Akt by 1,2-NQ was suppressed by pretreatment with tyrphostinA25, a specific EGFR tyrosine kinase inhibitor. Moreover, 1,2-NQ exerted the cytoprotective effect against for serum deprivation-induced cell death through the activation of EGFR-Akt signaling.

[Conclusions] These findings showed that 1,2-NQ directly activates EGFR-Akt signaling via chemical modification of EGFR.

DOI： 10.1254/jpssuppl.94.0_2-y-e1-3

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.

DOI： 10.1016/j.bbrc.2020.02.011

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Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

DOI： 10.15252/embj.2018100999

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The unfolded protein response (UPR) is activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which is called ER stress. ER stress sensors PERK, IRE1, and ATF6 play a central role in the initiation and regulation of the UPR; they inhibit novel protein synthesis and upregulate ER chaperones, such as protein disulfide isomerase, to remove unfolded proteins. However, when recovery from ER stress is difficult, the UPR pathway is activated to eliminate unhealthy cells. This signaling transition is the key event of many human diseases. However, the precise mechanisms are largely unknown. Intriguingly, reactive electrophilic species (RES), which exist in the environment or are produced through cellular metabolism, have been identified as a key player of this transition. In this review, we focused on the function of representative RES: nitric oxide (NO) as a gaseous RES, 4-hydroxynonenal (HNE) as a lipid RES, and methylmercury (MeHg) as an environmental organic compound RES, to outline the relationship between ER stress and RES. Modulation by RES might be a target for the development of next-generation therapy for ER stress-associated diseases.

DOI： 10.3390/ijms20071783

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Nitric oxide (NO) is a key signaling molecule that has various effects via S-nitrosylation, a reversible post-translational modification that affects the enzymatic activity, localization, and metabolism of target proteins. As chronic nitrosative stress correlates with neurodegeneration, the targets have received focused attention. Macrophage migration inhibitory factor (MIF) plays a pivotal role in the induction of gene expression to control inflammatory responses. MIF acts as a ligand for CD74 receptor and activates the Src-p38 mitogen-activated protein kinase (MAPK) cascade. MIF also elevates the expression of brain-derived neurotrophic factor (BDNF), which contributes to the viability of neurons. Here, we show that MIF is S-nitrosylated by a physiological NO donor. Interestingly, the induction of S-nitrosylation resulted in a loss of MIF activity following stimulation of the Src and p38 MAPK signaling pathways and the induction of BDNF expression. Our results shed light on the pathogenic mechanisms of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

DOI： 10.1248/bpb.b19-00025

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Amyloid-β precursor protein (APP) correlates with the pathogenesis of certain brain diseases, such as Alzheimer disease (AD). APP is cleaved by several enzymes to produce APP metabolites, including the amyloid beta peptide (Aβ), which accumulates in the brain of AD patients. However, the exact functions of APP metabolites remain elusive. In this study, using genome editing technology, we mutated juxta- and intra-membrane domains of murine APP in the mouse neuroblastoma cell line, Neuro2a. We identified several clones that expressed characteristic patterns of APP metabolites. Mutations in juxta- (deletion 673A), and intra-membrane (deletion 705-6LM) domains of APP, decreased overall levels of APP metabolites or decreased the level of α-secretase-cleaved carboxy-terminal fragment (αCTF), respectively. APP is known to influence neuronal differentiation; therefore, we used theses clones to dissect the function of APP metabolites during neuronal differentiation. One clone (CA), which expressed reduced levels of both FL-APP and αCTF, showed increased expression of the neuronal marker, β3-tubulin, and enhanced retinoic acid (RA)-induced neurite outgrowth. In contrast, a clone that expressed FL-APP, but was devoid of αCTF (CE), showed comparable expression of β3-tubulin and neurite outgrowth compared with normal Neuro2a cells. These data indicate that FL-APP is a suppressor of neurite outgrowth. Our data suggest a novel regulatory function of juxta- and intra-membrane domains on the metabolism and function of APP.

DOI： 10.1016/j.bbrc.2018.05.102

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Although the aggregation of amyloid-β peptide (Aβ) clearly plays a central role in the pathogenesis of Alzheimer's disease (AD), endosomal traffic dysfunction is considered to precede Aβ aggregation and trigger AD pathogenesis. A body of evidence suggests that the β-carboxyl-terminal fragment (βCTF) of amyloid-β precursor protein (APP), which is the direct precursor of Aβ, accumulates in endosomes and causes vesicular traffic impairment. However, the mechanism underlying this impairment remains unclear. Here we identified TMEM30A as a candidate partner for βCTF. TMEM30A is a subcomponent of lipid flippase that translocates phospholipids from the outer to the inner leaflet of the lipid bilayer. TMEM30A physically interacts with βCTF in endosomes and may impair vesicular traffic, leading to abnormally enlarged endosomes. APP traffic is also concomitantly impaired, resulting in the accumulation of APP-CTFs, including βCTF. In addition, we found that expressed BACE1 accumulated in enlarged endosomes and increased Aβ production. Our data suggested that TMEM30A is involved in βCTF-dependent endosome abnormalities that are related to Aβ overproduction.

DOI： 10.1371/journal.pone.0200988

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Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)-Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ-S-1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN-Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

DOI： 10.1038/s41598-017-04590-z

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Methylmercury (MeHg) results in cell death through endoplasmic reticulum (ER) stress. Previously, we reported that MeHg induces S-mercuration at cysteine 383 or 386 in protein disulfide isomerase (PDI), and this modification induces the loss of enzymatic activity. Because PDI is a key enzyme for the maturation of nascent protein harboring a disulfide bond, the disruption in PDI function by MeHg results in ER stress via the accumulation of misfolded proteins. However, the effects of MeHg on unfolded protein response (UPR) sensors and their signaling remain unclear. In the present study, we show that UPR is regulated by MeHg. We found that MeHg specifically attenuated inositol-requiring enzyme 1α (IRE1α)-x-box binding protein 1 (XBP1) branch, but not the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcriptional factor 6 (ATF6) branches. Treatment with GSK2606414, a specific PERK inhibitor, significantly inhibited MeHg-induced cell death. These findings suggest that MeHg exquisitely regulates UPR signaling involved in cell death.

DOI： 10.1248/bpb.b17-00359

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Specialist oncology pharmacists are being trained in Japan to assist cancer treatment teams. These specialized pharmacists address patients' physical and mental problems in pharmacist-managed cancer care clinics, actively participate in formulating treatment policies, and are beneficial in offering qualitative improvements to patient services and team medical care. However, the effect of outpatient treatment by oncology pharmacists on therapeutic outcomes and medical costs is still unknown. A retroactive comparative analysis of the treatment details and clinical course was conducted among three groups of patients: patients who underwent adjuvant chemotherapy managed by a gynecologic oncologist only (S arm), patients managed by a non-oncologist (general practice gynecologist) only (NS arm), and patients managed by both a non-oncologist and a specialist oncology pharmacist (NS+Ph arm). The medical cost per course was significantly lower for patients in the NS+Ph arm than for those in the other two arms. Surprisingly, the outpatient treatment rate in the NS+Ph arm was overwhelmingly high. The involvement of an oncology pharmacist did not make a significant difference in therapeutic outcomes such as recurrence rate and survival. The participation of oncology pharmacists in the management of cancer patients undergoing chemotherapy enables safe outpatient treatment and also reduces medical costs.

DOI： 10.1248/bpb.b17-00501

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Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

DOI： 10.1038/srep28944

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Nitric oxide (NO) plays a pivotal function in neurotransmission, vasodilation, proliferation, and apoptosis in various types of cells via protein S-nitrosylation. Previously we demonstrated that protein disulfide isomerase (PDI) is S-nitrosylated in brains manifesting sporadic neurodegenerative diseases. This modification results in dysfunction of its enzymatic activity and consequently the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). The aim of this study was to clarify the detailed function of NO on unfolded protein response (UPR) branches. We here found that the ER stress sensor IRE1α is S-nitrosylated. Interestingly, NO specifically abrogates ribonuclease activity, but not oligomerization or autophosphorylation of IRE1α. Site-directed mutagenesis revealed that Cys 931 and Cys951 in IRE1 are targets for S-nitrosylation. These mutants expressing in IRE1α knockout MEF showed a resistant role to the inhibition of nuclease activity by NO. Thus, we elucidated the effects of S-nitrosylation on ER stress sensors that mediate the UPR, and thus contribute to cell death pathways.

DOI： 10.1248/yakushi.15-00292-1

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Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.

DOI： 10.1038/srep14812

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Methylmercury (MeHg), an environmental pollutant, causes neuronal death via endoplasmic reticulum (ER) stress; however, the precise mechanism is not fully understood. The aim of this study was to elucidate the possible mechanism of MeHg-induced neurotoxicity. Treatment with MeHg resulted in a loss of cell viability in a concentration-dependent manner accompanying the expression of ER stress marker genes in human neuroblastoma SH-SY5Y cells. We next attempted to identify a target protein for MeHg in the ER. MeHg covalently modified protein disulfide isomerase (PDI), which is important for disulfide bond formation in nascent proteins in the ER lumen. S-Nitrosylation of the catalytic domains of PDI by nitric oxide was attenuated up to 50 % by a MeHg challenge in cells. The MeHg-modified C-terminal catalytic domain in PDI was detected by MALDI-TOF/MS. Furthermore, treatment with MeHg significantly attenuated the enzymatic activity of PDI. Taken together, these observations suggest that MeHg results in ER stress and following the unfolded protein response pathway via ER dysfunction due to S-mercuration of the C-terminus of PDI.

DOI： 10.1007/s12640-014-9494-8

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Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

DOI： 10.1016/j.bbrc.2014.11.066

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Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

DOI： 10.1248/bpb.b15-00364

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It has been known that reactive oxygen species (ROS) control the enzymatic and transcriptional activity of proteins via direct modification of cysteine residues. Hence, oxidation of cysteine thiol could be a vital modulator of signal transduction pathways. These findings indicate that some proteins serve as the sensor proteins highly sensitive to ROS. In this review, I show the relationship between intracellular ROS sensor and the regulation of protein function via oxidation.

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S-nitrosylation is a well-characterized reaction involving the covalent binding of nitric oxide (NO) to cysteine residues (Cys) in a protein. Similar to protein phosphorylation, S-nitrosylation is a post-translational modification involved in the regulation of a large number of intracellular functions and signaling events. Moreover, like phosphorylation, S-nitrosylation is precisely regulated in time and space. A procedure known as the biotin-switch method that specifically detects S-nitrosylated proteins (SNO-P) was recently developed by Snyder's group. They found that many proteins are substrates for NO, and several groups have attempted to identify other SNO-P by improving this method. In this review, we describe the SNO-P identified using modified versions of the biotin-switch method.

DOI： 10.1016/j.niox.2010.11.002

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Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.

DOI： 10.1073/pnas.1103503108

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The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.

DOI： 10.1016/j.bbrc.2011.04.103

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Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.

DOI： 10.1074/jbc.M110.190678

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PURPOSE: The present study was undertaken to clarify the effects of (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), a metabotropic glutamate receptor (mGluR) 1 antagonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), a mGluR2/3 agonist, and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), a mGluR4/8 agonist, on pentetrazol-induced kindled seizures. METHODS: Mice were anesthetized with pentobarbital; the electrodes and guide cannula were chronically implanted into the cortex and lateral ventricle. To induce kindling, pentetrazol at a dose of 40 mg/kg was injected once every 48 h. Behavioral and electroencephalographic seizures were monitored for 20 min following pentetrazol administration. Fully kindled mice were used for pharmacologic studies. RESULTS: Intracerebroventricular injection of AIDA and L-AP4 showed significant inhibitory effects on pentetrazol-induced kindled seizures. In addition, simultaneous use of AIDA and (2R,4R)-APDC or L-AP4 caused more potent inhibition of seizure activities. The inhibitory effect of AIDA on pentetrazol-induced kindled seizures was antagonized by (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG), a group I mGluR agonist; (2S)-a-ethylglutamic acid (EGLU), a group II mGluR antagonist; and (RS)-α-methyl-4-phosphonophenylglycine (MPPG), a group III mGluR antagonist. On the other hand, the inhibitory effect of L-AP4 was antagonized only by MPPG. DISCUSSION: It is proposed that mGluR1 antagonists and mGluR4/8 agonists show anticonvulsive effects on pentetrazol-induced kindled seizures. Furthermore, it is also proposed that the simultaneous use of an mGluR1 antagonist and an mGluR2/3 or mGluR4/8 agonist is a potential novel therapeutic strategy in epileptic disorders.

DOI： 10.1111/j.1528-1167.2010.02764.x

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We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.

DOI： 10.1111/j.1460-9568.2010.07160.x

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Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a well-defined chemokine implicated in the pathology of various neurodegenerative diseases and brain injuries, such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic injury. We investigated the effect of the activation of P2 purinoceptors on MCP-1 production in rat corticostriatal slice cultures. Treatment with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), a hydrolysis-resistant adenosine triphosphate (ATP) analog, induced MCP-1 production in astrocytes. The induction was in a concentration-dependent manner and was antagonized by a P2 purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. The inhibition of an extracellular signal-regulated kinase (ERK) pathway by PD98059 and U0126 significantly suppressed ATPgammaS-induced MCP-1 mRNA expression and protein production, while inhibition of c-Jun N-terminal kinase by SP600125 resulted in the partial suppression. Conversely, SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, significantly enhanced ATPgammaS-induced MCP-1 production. Similar effects of ERK and p38 MAP kinase inhibitors on MCP-1 production were observed in the slices stimulated by ATP and BzATP. These results demonstrate that astrocytic MCP-1 production induced by P2 purinoceptor stimulation is reciprocally regulated by ERK and p38 MAP kinases in the organotypic slice cultures.

DOI： 10.1002/jnr.21982

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Accumulating evidence indicates a pivotal role for neuroinflammation in ischemic and excitotoxic brain injury. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is a CXC chemokine implicated in the infiltration of inflammatory cells into the brain parenchyma. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA)-induced neuronal injury on CINC-1 production in the organotypic cortico-striatal slice cultures. Treatment with 50 microM NMDA for 3 - 4 h caused devastating neuronal damage and increased CINC-1 production. Immunohistochemical analysis revealed that the CINC-1 immunoreactivity was predominantly detected in astrocytes. NMDA failed to induce CINC-1 production in enriched astrocyte cultures or neuron-depleted slice cultures, suggesting that NMDA acted on neuronal cells to induce astrocytic CINC-1 production. NMDA-induced CINC-1 mRNA expression was significantly inhibited by U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. These results suggest that NMDA-evoked neuronal injury induced astrocytic CINC-1 production via a MEK/ERK signaling pathway. Manipulation of this signaling pathway may serve as a target for suppressing neuroinflammation and, thereby, treating ischemic brain injury.

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The interaction of glucocorticoid modulatory element-binding protein 1 (GMEB1) with procaspase-2, -8, or -9 prevents caspase oligomerization and maturation. In the present study, we examined the effect of GMEB1 on neuronal apoptosis induced by hypoxia and oxidative stress. GMEB1 effectively attenuated caspase activation and apoptosis caused by these stresses in human neuroblastoma SK-N-MC cells, indicating that it functions as a potent inhibitor of caspase activation and apoptosis in response to oxidative stress. We propose that GMEB1 blocks pro-apoptosis signals induced by a variety of stresses.

DOI： 10.1016/j.neulet.2008.04.023

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Protein quality control is a critical feature of intracellular homeostasis. In particular, unfolded or misfolded proteins resulting from environmental stresses or free radicals are rapidly degraded via the ubiquitin-proteasome pathway. Nitric oxide (NO), a free radical gas, has been reported to be involved in such processes as vasorelaxation and neurotransmission. Conversely, NO also is implicated in neuronal cell death or neurodegeneration. Recent reports suggest that S-nitrosylation of proteins is a significant cause of neural dysfunction leading to neurodegenerative disorders. Specifically, S-nitrosylation of parkin eventually leads to the accumulation of unfolded proteins and subsequent neuronal death. The focus of this review is the identity of the target of NO. Nitrosative stress prevents normal functioning of the endoplasmic reticulum (ER) via S-nitrosylation of protein-disulfide isomerase (PDI), which is located in the ER lumen. This may contribute to the accumulation of misfolded proteins, as well as sustained activation of the unfolded protein response (UPR) pathway. These phenomena may be linked to the development of sporadic neurodegenerative diseases.

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Using a conditioned place paradigm, we examined the involvement of the bed nucleus of the stria terminalis (BST) in the negative affective component of visceral and somatic pain induced by intraperitoneal acetic acid and intraplantar formalin injections, respectively, in rats. Bilateral BST lesions suppressed both the acetic acid- and formalin-induced conditioned place aversion, suggesting the crucial role of the BST in the negative affective component of visceral and somatic pain.

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It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.

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Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.

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Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.

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Caspases are divided into two classes: initiator caspases, which include caspase-8 and -9 and possess long prodomains, and effector caspases, which include caspase-3 and -7 and possess short prodomains. Recently, we demonstrated that glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the prodomain of procaspase-2, thereby disrupting its autoactivation and the induction of apoptosis. Here we show that GMEB1 is also capable of binding to procaspase-8 and -9. GMEB1 attenuated the Fas-mediated activation of these caspases and the subsequent apoptosis. The knockdown of endogenous GMEB1 using RNA interference revealed that cells with decreased GMEB1 expression are more sensitive to stress and undergo accelerated apoptosis. Transgenic mice expressing a neurospecific GMEB1 had smaller cerebral infarcts and less brain swelling than wild-type mice in response to transient focal ischemia. These results suggest that GMEB1 is an endogenous regulator that selectively binds to initiator procaspases and inhibits caspase-induced apoptosis.

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The inhibitory effects of astrapterocarpan, formononetin, and calycosin isolated from Astragalus membraneceus on platelet-derived growth factor (PDGF)-BB-induced proliferative response in rat vascular smooth muscle cells (A10 cells) were investigated. Astrapterocarpan significantly inhibited PDGF-BB-induced cell proliferation and DNA synthesis in a concentration-dependent manner. This inhibition was not attributed to toxicity. In contrast, formononetin and calycosin had no effect. We next examined the effect of astrapterocarpan on PDGF-BB signal transduction. Astrapterocarpan inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERIC1/2) mitogen-activated protein (MAP) kinase. However, this compound had no effect on phosphorylation of PDGF-beta-receptor, Akt kinase and p38 MAP kinase. These results indicated that astrapterocarpan inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and that this effect may be mediated, at least in part, by inhibition of the ERK1/2 MAP kinase cascade.

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To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.

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Caspases are the primary executioners of apoptosis. Although procaspases believe to exist as inactive forms in cells, the detailed regulatory system remains unclear. Here we show that glucocorticoid modulatory element-binding protein 1 (GMEB1) is capable of binding to the prodomain of caspase-2. We found that this molecule inhibits the autoproteolytic activation of procaspase-2 by oligomerization on a chemical compound-dependent system. These findings indicated that GMEB1 might be an endogenous inhibitory protein that selectively interacts with prodomain of caspase-2 to disrupt the autoactivation.

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Neurons are highly vulnerable to ischemic/hypoxic stress, while glial cells show tolerance to such stress. However, the mechanisms for tolerance acquisition in glial cells have yet to be established. We attempted to isolate and identify a stress protein that is upregulated in response to hypoxia in human astrocytoma CCF-STTG1 cells. In particular, pyruvate kinase (PK) was upregulated by hypoxia in CCF-STTG1 cells. Hypoxia-inducible factor 1 (HIF-1), the primary transcription factor that is responsible for multiple gene activation under hypoxia, plays a critical role in PK expression during hypoxic challenge. To determine whether newly synthesized PK is involved in tolerance to hypoxic stress, we established the PK-overexpressing neuronal cells. Overexpression of the wild-type, but not the kinase-negative mutant, resulted in attenuation of the loss of cell viability and the typical apoptotic features by hypoxia or oxidative stress in SK-N-MC cells. These findings suggest that upregulation of PK may result in acquisition of tolerance against hypoxic stress, and that the antioxidant effect may be involved in the protective effect of PK.

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Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

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NF (nuclear factor)-kappaB is known to be a critical transcription factor in inflammatory responses. We have reported that herbimycin A, a potent Src tyrosine kinase inhibitor, attenuates the NF-kappaB activation triggered by cytokines, bacterial endotoxin, and hydrogen peroxide. Accompanying the suppression by this agent, NF-kappaB-dependent gene expressions, such as cytokine, chemokine, and inducible-type nitric oxide, are specifically inhibited in glial cells. In the present study, we attempted to elucidate the possible target protein for herbimycin A on this pathway. We demonstrate here that herbimycin A preferentially inhibits IKK (IkappaB kinase)beta. Furthermore, substituting alanine for the cysteine at 59 (Cys59) in IKKbeta resulted in the insensitivity to herbimycin A, suggesting that this compound may interact with the Cys59 residue located near the catalytic ATP binding site. Taken together, these results indicate that herbimycin A can be considered a novel candidate for an anti-inflammatory drug agent through its specific inhibition of IKKbeta, which results in prevention of the expression of NF-kappaB-dependent genes implicated in the pathogenesis of inflammatory responses.

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Mammalian cells acquire tolerance against multiple stressors through the high-level expression of stress-responsible genes. We have previously demonstrated that protein-disulfide isomerase (PDI) together with ubiquilin are up-regulated in response to hypoxia/brain ischemia, and play critical roles in resistance to these damages. We show here that ubiquilin interacts preferentially with poly-ubiquitin chains and 19S proteasome subunits. Taken together, these results suggest that ubiquitin could serve as an adaptor protein that both interacts with PDI and mediates the delivery of poly-ubiquitylated proteins to the proteasome in the cytosol in the vicinity of the endoplasmic reticulum membrane.

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Transient forebrain ischemia induces a delayed neuronal death in the CA1 area of the hippocampus. However, the mechanism leading to this phenomenon has yet to be established. The authors used an mRNA differential-display method to isolate genes for which mRNA levels change only in the hippocampus during ischemia/reperfusion. They succeeded in identifying the product of one down-regulated gene as phosphatidylinositol 4-kinase (PI 4-K). Compared with control levels, PI 4-K mRNA expression in the hippocampus, but not the cerebral cortex, was significantly decreased by 30% and about 80% 1 and 7 days after ischemia/reperfusion, respectively. Interestingly, PI 4-K and PI bisphosphate levels were selectively decreased in the CA1 region, but not other regions, whereas TUNEL-positive cells could be detected 3 days after ischemia. Consistent with these results, PI 4-K expression was suppressed by hypoxia in SK-N-MC neuroblastoma cells before loss of cell viability. Overexpression of wild-type PI 4-K, but not the kinase-negative mutant of PI 4-K (K1789A), recovered the loss of viability induced by hypoxia. These findings strongly suggest that a prior decrease in PI 4-K and PI bisphosphate levels caused by brain ischemia/hypoxia is partly involved in delayed neuronal cell death.

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Up-regulation of several stress proteins such as heat-shock proteins and glucose-regulated proteins participate in tolerance against environmental stress. Previously, we found that protein-disulfide isomerase (PDI) is specifically up-regulated in response to hypoxia/brain ischemia in astrocytes. In addition, the overexpression of this gene into neurons protects against apoptotic cell death induced by hypoxia/brain ischemia. To address the detailed function of PDI, we screened for proteins that interact with PDI using the yeast two-hybrid system. We report here that PDI interacts with ubiquilin, which has a ubiquitin-like domain and a ubiquitin-associated domain. Interestingly, ubiquilin is also up-regulated in response to hypoxia in glial cells with a time course similar to that of PDI induction. In hypoxia-treated glial cells, the endogenous ubiquilin and PDI were almost completely co-localized, suggesting that ubiquilin is an endoplasmic reticulum-associated protein. Overexpression of this gene in neuronal cells resulted in significant inhibition of the DNA fragmentation triggered by hypoxia, but not that induced by nitric oxide or staurosporine. Moreover, ubiquilin has the ability to attenuate CHOP induction by hypoxia. These observations suggested that ubiquilin together with PDI have critical functions as regulatory proteins for CHOP-mediated cell death, and therefore up-regulation of these proteins may result in acquisition of tolerance against ischemic stress in glial cells.

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It has been demonstrated from studies using NF-kappa B inhibitors that NF-kappa B may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the actions of the inhibitors are less selective and highly cytotoxic, We constructed stable clones of C6 cells transfected with two types of I kappa B alpha mutant genes (I kappa B alpha(SS-->AA); Ser-32/36 to Ala-32/36, I kappa B alpha(KK-->RR);Lys-21/22 to Arg-21/22). I kappa B alpha(SS-->AA) strongly inhibited (1) LPS-, IL-1 beta-, and TNF-alpha-induced nuclear translocation and DNA binding of NF-kappa B to the kappa B site; and (2) iNOS induction stimulated by LPS or IL-1 beta plus IFN-gamma, These results indicate that NF-kappa B plays a critical role in cytokines and/or LPS-induced iNOS induction. Surprisingly, similar to the endogenous I kappa B alpha, I kappa B alpha(KK-->RR) was degraded by various stimuli, and proteasome inhibitors blocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced I kappa B alpha degradation by the ubiquitin-proteasome pathway. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3293

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Members of the caspase family are essential executors of apoptosis, Caspase-2 has two messenger RNAs generated by alternative splicing, which encode caspase-2L and caspase-2S, Although caspase-2L induces apoptosis, caspase-2S also has the ability to antagonize cell death. Experiments in caspase-2-deficient mice showed that caspase-2 functions to delay cell death in some neuronal populations, suggesting that caspase-2S dominantly acts for cell survival in the brain, However, the mechanism of caspase-2S-mediated anti-apoptotic effect is still unclear. Here. we isolated a protein that interacts with caspase-2S, designated as Ich-1S (caspase-2S)-binding protein (ISBP), by yeast two-hybrid screening using full-length caspase-2S cDNA as a bait. ISBP is identical to the recently isolated calcium and integrin-binding protein, and a small molecule calcium-binding protein with two EF-hand motifs of its C-terminus. In vitro transcribed and translated ISBP interacts specifically with glutathione-S-transferase-fused caspase-2S, Moreover, the interaction between ISBP and caspase-2S was observed in cultured cells, Northern blot analysis indicated that ISBP may be a ubiquitous protein. Interestingly, ISBP can partially inhibit the processing of pro-caspase-2L induced by anti-Fas antibody-treated Jurkat cytosolic lysates, These results suggested that ISBP mag be the mediator for the survival function of caspase-2S. (C) 2000 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(00)01351-X

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DOI： 10.1016/S0167-4889(99)00131-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor kappa B (NF-kappa B)-dependent manner. In this study, we focused further on the regulation of NF-kappa B, The activation of NF-kappa B and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1 beta (IL-1 beta) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-kappa B into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of I kappa B alpha, a regulatory protein of NF-kappa B, Nascent I kappa B alpha began to degrade 5 min after treatment with IL-1 beta and disappeared completely after 15 min. However, I kappa B alpha returned to basal levels after 45-60 min. Interestingly, resynthesized I kappa B alpha was already phosphorylated at Ser-32, These results suggest that 1) the upstream signals are still activated, although the translocation of NF-kappa B peaks at 15 min; and 2) the regulated protein(s) acts downstream of I kappa B alpha phosphorylation, Western blotting showed that the resynthesized and phosphorylated I kappa B molecules were also upward-shifted by multi-ubiquitination in response to IL-1 beta treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1 beta for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated I kappa B alpha, These results indicate that the transient translocation of NF-kappa B in response to IL-1 beta may be partly dependent on transient proteasome activation.

DOI： 10.1074/jbc.274.22.15875

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The aim of this study was to elucidate the possible mechanism of HSP induction in response to hypoxia in rat primary astrocytes. Treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the increase in HSP70 in a concentration-dependent manner. p38 MAPK was activated in response to hypoxic treatment. These results suggest that p38 MAPK positively regulates hypoxia-induced HSP70 expression in astrocytes. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)01178-6

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Language：Japanese   Publisher：The Japanese Pharmacological Society  

To elucidate the intracellular mechanism of NF-κB activation, we performed the involvement of IκBα of NF-κB in the expression of inducible NO synthase (iNOS) and chemokine (CINC) following pretreatment with bacterial endotoxin (LPS) or IL-1β, respectively, using rat C6 glioma cells. We found that herbimycin A, a tyrosine protein kinase inhibitor, blocked: 1) LPS/IFNγ-induced iNOS expression, 2) LPS-induced intranuclear translocation of activated NF-κB (p50·p65) and 3) IFNγ-induced autophosphorylation and activation of Jak 2 and Stat 1 as well as intranuclear translocation of phosphorylated Stat 1. Furthermore, transfection of a dominant negative form of IκBα(SS→AA) suppressed LPS/IFNγ-induced iNOS expression, suggesting that NF-κB, in particular, IκBα molecules could play important roles in the iNOS expression. We also found in IL-1β-induced CINC expression using cultured C6 glioma cells, the transient translocation of NF-κB in response to IL-1β is partly dependent on transient proteasome activation. Thus we suggest that the formation of heterodimer p50·p65 from inactive trimer p50·p65·IκBα particularly, proteolytic degradation and dissociation of IκBα from p50·p65 are a critical phase in NF-κB activation during LPS-induced iNOS and IL-1β-induced CINC expression in astroglial cells.

DOI： 10.1254/fpj.114.supplement_92

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT-RAVEN PUBL  

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. When the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effects were also inhibited by pretreatment with staurosporine or calphostin C. In addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+](i) were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m(3) mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.

DOI： 10.1046/j.1471-4159.1996.67010131.x

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Insulin/dexamethasone/methylisobutylxanthine (hormones/IBMX) induce 3T3-L1 fibroblasts to differentiate into adipocytes. Our previous study suggested that pertussis toxin (IAP)-sensitive GTP-binding protein(s) (G-protein) is involved in the process of differentiation by hormones/IBMX, accompanied by c-fos induction. Northern blotting indicated that among the IAP-sensitive G-proteins, the levels of Gi2α, Goα, and Gi3α mRNA were decreased, increased and unchanged, respectively. Gi1α was undetectable and IAP attenuated the decrease in Gi2α mRNA level but did not affect the change in Goα mRNA level during the adipocyte differentiation. These results indicate that IAP-sensitive Gi2α mRNA level is decreased during adipocyte differentiation. A combination of phosphatidylinositol-specific phospholipase C (PI-PLC) and IBMX induced c-fos expression in 3T3-L1 fibroblasts similar to that induced with hormones/IBMX. c-fos induced by both stimulators was also diminished by anti-inositolglycan antibody or anti-PI-PLC antiserum. Insulin stimulated the release of inositolproteoglycan and diacylglycerol from 3T3-L1 fibroblasts, which was suppressed by IAP treatment. These findings suggested that one of the pathways of adipocyte differentiation induced by hormones/IBMX occurs via the inositolglycan-specific PI-PLC cascade coupled to IAP-sensitive G-protein(s). Both activation of glycerophosphate dehydrogenase and stimulation of insulin-dependent 2-deoxyglucose uptake induced by hormones/IBMX were enhanced in protein kinase C-depleted cells exposed to phorbol 12-myristate 13-acetate (PMA), and attenuated in IAP-treated cells. The level of a 32P-labeled 52 kDa protein in plasma membrane fractions immunoprecipitated by anti-PI-PLC antiserum was increased by PMA stimulation, abolished in PMA-treated cells, and increased in IAP-treated cells. These findings suggest that protein kinase C phosphorylates PI-PLC, resulting in a decrease in PI-PLC activity related to the signal transduction pathway of adipocyte differentiation of 3T3-L1 fibroblasts. © 1994.

DOI： 10.1016/0167-4889(94)90204-6

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.niox.2010.05.028

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.839

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Other Link： http://search.jamas.or.jp/link/ui/2007306169

Language：Japanese   Publisher：共立出版(株)  

最近孤発性パーキンソン病患者の脳内でParkinがニトロシル化されており,ストレスにより活性が抑制されるとの報告があった.以前より孤発性パーキンソン病で指摘されていた酸化ストレス因子によるParkinの活性低下が,孤発性パーキンソン病の原因の1つである可能性もでてきた.また,Parkinの活性制御についてはいまだ不明な点が多いが,活性制御のメカニズムの解明は孤発性パーキンソン病の病因の解明・治療に役立つものと思われる(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publisher：ELSEVIER SCIENCE BV  

We have attempted to elucidate the mechanism of apoptotic cell death induced by hypoxia (very low oxygen conditions) in neuronal cells, Human neuroblastoma SK-N-MC cells under hypoxic conditions resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent chromatin dye. Pretreatment with Z-Asp-CH2-DCB, a caspase inhibitor, suppressed the DNA ladder in response to hypoxia in a concentration-dependent manner. An increase in caspase-3-like protease (DEVDase) activity,vas observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm the involvement of caspase-3 during apoptosis, Western blot analysis mas performed using anti-caspase-3 antibody. The 20- and 17-kDa proteins, corresponding to the active products of caspase-3, were generated in hypoxia-challenged lysates in which processing of the full length form of caspase-3 was evident. With a time course similar to this caspase-3 activation, hypoxic stress caused the cleavage of PARP, yielding an 85-kDa fragment typical of caspase activity. In addition, caspase-2 was also activated by hypoxia, and the stress elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that caspase activation and cytochrome c release play roles in hypoxia-induced neuronal apoptosis. (C) 1998 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(98)01363-5

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant-negative form of c-Ha-Ras (Asn-17) abrogated IFN-gamma-induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21(ras) and MEK1 are required for IFN-gamma-induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras-MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn-17 Ras expression nor concentrations of PD98059, which completely abrogated IFN-gamma-induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras-MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)00383-9

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Herbimycin A, a potent tyrosine kinase inhibitor, suppressed nitric oxide synthase (NOS) induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in C6 glial cells, LPS activated NF-kappa B, and this effect was inhibited by pretreatment with herbimycin A, In addition, IFN-gamma activated the tyrosine protein kinase, JAK2, and tyrosine-phosphorylation by itself was also inhibited by herbimycin A. These results suggest that herbimycin A suppresses iNOS induction by inhibition of both NF-kappa B activation caused by LPS, and tyrosine-phosphorylation of JAK2 caused by IFN-gamma in C6 glioma cells.

DOI： 10.1016/0014-5793(95)00933-Z

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