Hirokazu Hara

Publications of Hirokazu Hara

• TPA induces the expression of EC-SOD in human monocytic THP-1 cells: Involvement of PKC, MEK/ERK and NOX-derived ROS.

  Authors: Junya Makino, Tetsuro Kamiya, Hirokazu Hara, Tetsuo Adachi

  Free radical research. 03/2012; 46(5):637-44.

  Abstract Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributesAbstract Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.

• Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability.

  Authors: Tetsuo Adachi, Mayumi Teramachi, Hiroyuki Yasuda, Tetsuro Kamiya, Hirokazu Hara

  Archives of biochemistry and biophysics. 01/2012; 520(1):30-5.

  Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairmentDiabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC-dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC-dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.

• Endoplasmic reticulum stress induces retinal endothelial permeability of extracellular-superoxide dismutase.

  Authors: Tetsuo Adachi, Hiroyuki Yasuda, Shinsuke Nakamura, Tetsuro Kamiya, Hirokazu Hara, Hideaki Hara, Tsunehiko Ikeda

  Free radical research. 09/2011; 45(9):1083-92.

  The aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by theThe aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by the investigation of two possibilities: first, change of EC-SOD expression in the retina; and secondly, leakage of EC-SOD through the endothelial monolayer by the treatment with endoplasmic reticulum (ER) stress inducers because ER stress is known to be involved in the vascular impairment in diabetic retinopathy. Intravitreous injection of tunicamycin in mice increased the permeability of tracer dye across retinal blood vessels while the retinal EC-SOD mRNA level was not changed. The leakage of EC-SOD through the retinal endothelial cell layer was elevated by the treatment with thapsigargin or tunicamycin. The expression of claudin-5 was significantly decreased by the treatment with the ER stress inducers. These phenomena were significantly suppressed by the pre-treatment of endothelial cells with a chemical chaperone 4-phenylbutyric acid. Our observations suggest that ER stress leads to the down-regulation of claudin-5 among tight junction proteins and may induce the elevation of endothelial permeability and leakage of EC-SOD into the vitreous body.

• Inhibitory effects of chalcone glycosides isolated from Brassica rapa L. 'hidabeni' and their synthetic derivatives on LPS-induced NO production in microglia.

  Authors: Hirokazu Hara, Yoko Nakamura, Masayuki Ninomiya, Ryosuke Mochizuki, Tetsuro Kamiya, Elias Aizenman, Mamoru Koketsu, Tetsuo Adachi

  Bioorganic & medicinal chemistry. 07/2011; 19(18):5559-68.

  Activation of microglia induces the production of various inflammatory mediators including nitric oxide (NO), leading to neurodegeneration in many central nervous system diseases. In this study, weActivation of microglia induces the production of various inflammatory mediators including nitric oxide (NO), leading to neurodegeneration in many central nervous system diseases. In this study, we examined the effects of chalcone glycosides isolated from Brassica rapa L. 'hidabeni' on lipopolysaccharide (LPS)-induced NO production using rat immortalized microglia HAPI cells. 4'-O-β-D-Glucopyranosyl-3',4-dimethoxychalcone (A2) inhibited LPS-induced inducible NO synthase (iNOS) expression and NO production. However, A2 did not affect nuclear factor-κB and mitogen-activated protein kinase pathways. The signal transduction and activator of transcription 1 (STAT1), which is activated via production of IFN-β by LPS, is an important transcription factor responsible for LPS-induced iNOS expression. A2 suppressed LPS-induced phosphorylation and nuclear translocation of STAT1, although it had no effects on LPS-induced IFN-β expression. These results indicate that the inhibitory effect of A2 is due to the prevention of STAT signaling. Moreover, structure-activity relationship studies on newly synthesized 'hidabeni' chalcone derivatives showed that 4'-O-β-D-glucopyranosyl-3'-methoxychalcone (A11), which has no functional groups in the B-ring, inhibits LPS-induced NO production more potently than A2.

• ER stress inducer, thapsigargin, decreases extracellular-superoxide dismutase through MEK/ERK signalling cascades in COS7 cells.

  Authors: Tetsuro Kamiya, Aya Obara, Hirokazu Hara, Naoki Inagaki, Tetsuo Adachi

  Free radical research. 03/2011; 45(6):692-8.

  It has been reported that tubular cells suffer an endoplasmic reticulum (ER) stress during the development of chronic kidney disease, which is a potent risk factor of cardiovascular disease.It has been reported that tubular cells suffer an endoplasmic reticulum (ER) stress during the development of chronic kidney disease, which is a potent risk factor of cardiovascular disease. Moreover, under these conditions, reactive oxygen species are generated and induce cell injury. Extracellular-superoxide dismutase (EC-SOD) is a member of SODs and protects the cells from oxidative stress. Here, it is demonstrated that thapsigargin, an ER stress inducer, decreased EC-SOD expression, whereas the expression of Cu,Zn-SOD and Mn-SOD was not changed. On the other hand, another ER stress inducer, tunicamycin, did not affect the expression of EC-SOD. Further, it was shown that thapsigargin has the ability to activate extracellular-signal regulated kinase (ERK), but tunicamycin does not. Moreover, pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK, suppressed thapsigargin-triggered EC-SOD reduction, suggesting that MEK/ERK signalling should play an important role in the regulation of EC-SOD in COS7 cells under ER stress conditions.

• Extracellular-superoxide dismutase expression in COS7 cells exposed to cadmium chloride.

  Authors: Aya Obara, Tetsuro Kamiya, Misato Izumi, Hirokazu Hara, Harutaka Yamada, Tetsuo Adachi

  Biological & pharmaceutical bulletin. 01/2011; 34(9):1443-7.

  Cadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactiveCadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactive oxygen species (ROS) and induces cytotoxicity. Extracellular-superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the cells from damaging effects of ROS; however, the effect of Cd on the expression of EC-SOD in COS7 cells remains unclear. In this study, exposure to cadmium chloride (CdCl₂) enhanced intracellular ROS generation and induced COS7 cell death. Moreover, exposure to Cd decreased the expression of EC-SOD at mRNA and protein levels, but not of other SOD isozymes, copper-and zinc-containing SOD and manganese-containing SOD. The reduction of EC-SOD and cell viability was partially attenuated by pretreatment with an antioxidant, N-acetylcysteine. Further, we determined the involvement of p38-mitogen-activated protein kinase (p38-MAPK) in the reduction of EC-SOD. From these observations, p38-MAPK signaling cascades activated by ROS play a pivotal role in the reduction of EC-SOD, and it is concluded that the reduction of EC-SOD leads to a decrease in the resistance to oxidative stress of Cd-exposed COS7 cells.

• Effect of hypoxia mimetic cobalt chloride on the expression of extracellular-superoxide dismutase in retinal pericytes.

  Authors: Tetsuo Adachi, Kazunari Aida, Hiroko Nishihara, Tetsuro Kamiya, Hirokazu Hara

  Biological & pharmaceutical bulletin. 01/2011; 34(8):1297-300.

  The initial clinical stage of diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. The increased formation of reactive oxygen species (ROS) isThe initial clinical stage of diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. The increased formation of reactive oxygen species (ROS) is thought to be a key event in the pathogenesis of DR. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that is distributed mainly in vascular cells and protects cells from ROS by scavenging superoxide anion. Treatment with cobalt chloride (CoCl(2)) decreased the expression of EC-SOD but not other SOD isozymes in pericytes accompanied with an increase of intracellular ROS production. Pre-treatment with N-acetylcysteine (NAC) significantly suppressed the ROS production and down-regulation of EC-SOD. We observed the activation of caspase-3 and DNA fragmentation as signs of apoptotic process by CoCl(2) treatment. In addition, these phenomena were significantly inhibited by pre-treatment with NAC. EC-SOD enhancer 4-phenyl butyric acid also suppressed the caspase-3 activation. It is known that the presence of a high level of EC-SOD throughout the vessel walls might have an important protective role against superoxide in the vascular system. The decrease in EC-SOD expression accompanied with elevation of ROS level in pericytes under hypoxia might induce and/or promote the ROS-triggered apoptosis of pericytes and the development of pathogenesis in DR.

• Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells.

  Authors: Tetsuro Kamiya, Junya Makino, Hirokazu Hara, Naoki Inagaki, Tetsuo Adachi

  Journal of cellular biochemistry. 11/2010; 112(1):244-55.

  Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It isLeukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.

• Endoplasmic reticulum stress inducers provide protection against 6-hydroxydopamine-induced cytotoxicity.

  Authors: Hirokazu Hara, Tetsuro Kamiya, Tetsuo Adachi

  Neurochemistry international. 10/2010; 58(1):35-43.

  6-Hydroxydopamine (6-OHDA) is a neurotoxin used to establish experimental models of Parkinson's disease. Exposure to 6-OHDA results in cell death associated with oxidative stress. Pretreatments with6-Hydroxydopamine (6-OHDA) is a neurotoxin used to establish experimental models of Parkinson's disease. Exposure to 6-OHDA results in cell death associated with oxidative stress. Pretreatments with sublethal oxidative stress and some pharmacological drugs have been shown to exert preconditioning effects on cytotoxicity caused by 6-OHDA. In this study, we investigated whether endoplasmic reticulum (ER) stress exerts preconditioning effects on 6-OHDA-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Pretreatment with ER stress inducers, thapsigargin (Tg) and tunicamycin (Tm), promoted GRP78 mRNA induction and ATF4 translation, which are ER stress markers, under our experimental conditions and protected against the cytotoxicity. The protective effect of Tg was more potent than that of Tm. We also found that Tg induced the expression of the antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner, whereas Tm had a weak effect on HO-1 induction. Flow cytometric analysis revealed that reactive oxygen species generated by 6-OHDA were more effectively suppressed in cells pretreated with Tg than with Tm. Therefore, it is likely that Tg enhances antioxidative defenses in SH-SY5Y cells compared with Tm. Because actinomycin D inhibited HO-1 induction by Tg, the induction of HO-1 may be regulated at the transcriptional level. Moreover, the specific eIF2α phosphatase inhibitor salubrinal augmented Tg-induced HO-1 expression. Therefore, the downstream signaling pathway of eIF2α might be involved in Tg-induced HO-1 expression. On the other hand, the reporter assay revealed that Tg stimulated the antioxidant response element (ARE) that is located in regulatory regions of antioxidant genes such as HO-1. Taken together, our data suggest that preconditioning effects induced by Tg mediate an adaptive response to 6-OHDA-induced cytotoxicity via phosphorylation of eIF2α and activation of the ARE.

• Allograft inflammatory factor-1 is overexpressed and induces fibroblast chemotaxis in the skin of sclerodermatous GVHD in a murine model.

  Authors: Aihiro Yamamoto, Eishi Ashihara, Yoko Nakagawa, Hiroshi Obayashi, Mitsuhiro Ohta, Hirokazu Hara, Tetsuo Adachi, Takahiro Seno, Masatoshi Kadoya, Masahide Hamaguchi, Hidetaka Ishino, Masataka Kohno, Taira Maekawa, Yutaka Kawahito

  Immunology letters. 10/2010; 135(1-2):144-50.

  Allograft inflammatory factor (AIF)-1 has been identified in chronic rejection of rat cardiac allografts and is thought to be involved in the immune response. We previously showed that AIF-1 wasAllograft inflammatory factor (AIF)-1 has been identified in chronic rejection of rat cardiac allografts and is thought to be involved in the immune response. We previously showed that AIF-1 was strongly expressed in synovial tissues in rheumatoid arthritis and that rAIF-1 increased the IL-6 production of synoviocytes and peripheral blood mononuclear cells. Recently, the expression of AIF-1 has been reported in systemic sclerosis (SSc) tissues, whose clinical features and histopathology are similar to those of chronic graft-vs-host disease (GVHD). To clarify the pathogenic mechanism of fibrosis, we examined the expression and function of AIF in sclerodermatous (Scl) GVHD mice. We demonstrated that immunoreactive AIF-1 and IL-6 were significantly expressed in infiltrating mononuclear cells and fibroblasts in thickened skin of Scl GVHD mice compared with control. The immunohistochemical findings were confirmed by Western blot analysis. Wound healing assay also revealed that rAIF-1 increased the migration of normal human dermal fibroblasts (NHDF) directly, but cell growth assay did not show that rAIF-1 increased the proliferation of them. These findings suggest that AIF-1, which can induce the migration of fibroblasts and the production of IL-6 in affected skin tissues, is an important molecule promoting fibrosis in GVHD. Although the biological function of AIF-1 has not been completely elucidated, AIF-1 can induce IL-6 secretion on mononuclear cells and fibroblast chemotaxis. AIF-1 may accordingly provide an attractive new target for antifibrotic therapy in SSc as well as Scl GVHD.

• Effect of pioglitazone on various parameters of insulin resistance including lipoprotein subclass according to particle size by a gel-permeation high-performance liquid chromatography in newly diagnosed patients with type 2 diabetes.

  Authors: Koji Nakano, Goji Hasegawa, Michiaki Fukui, Masahiro Yamasaki, Kiyoshi Ishihara, Tooru Takashima, Yoshihiro Kitagawa, Aya Fujinami, Mitsuhiro Ohta, Hirokazu Hara, Tetsuo Adachi, Masakazu Ogata, Hiroshi Obayashi, Naoto Nakamura

  Endocrine journal. 02/2010; 57(5):423-30.

  Pioglitazone is an insulin-sensitizing agent that has been reported to have anti-arteriosclerotic effects. The aim of this study was to obtain a better understanding of the mechanism involved in thePioglitazone is an insulin-sensitizing agent that has been reported to have anti-arteriosclerotic effects. The aim of this study was to obtain a better understanding of the mechanism involved in the insulin sensitizing effect of pioglitazone. A total of 50 newly diagnosed patients with type 2 diabetes were enrolled in this study and divided into two groups, 25 of who were treated with 15 mg/day pioglitazone and 25 with 500 mg/day metformin for 12 weeks. Changes in various parameters of insulin resistance including lipoprotein subclass according to particle size determined by high performance liquid chromatography, as well as glucose metabolism, were monitored to determine the relationship between lipoprotein subclass and other insulin resistance parameters. Both pioglitazone and metformin treatment were associated with significant reductions in hyperglycemia, HOMA-IR and HbA1c levels. Pioglitazone treatment, but not metformin treatment resulted in significant reductions in serum large very low-density lipoprotein (VLDL: 44.5-64.0 nm) and increases in serum adiponectin levels (both <0.001). In the pioglitazone group, the change in large VLDL levels correlated positively with changes in HbA1c (r=0.468, P=0.0174), HOMA-IR (r=0.593, P=0.0014), very small LDL (r=0.714, P<0.0001) and net electronegative charged modified-LDL (r=0.412, P=0.0399), and inversely with changes in adiponectin level (r=-0.526, P=0.0061). The results in this study suggest that the hypoglycemic effect of pioglitazone is achieved mainly through improvement of hepatic insulin resistance, and that pioglitazone may have an antiatherosclerotic effect by decreasing serum atherogenic modified-LDL and by increasing adiponectin.

• Regulation of extracellular-superoxide dismutase in rat retina pericytes.

  Authors: Tetsuo Adachi, Hiroyuki Yasuda, Kazunari Aida, Tetsuro Kamiya, Hirokazu Hara, Ken-ichi Hosoya, Tetsuya Terasaki, Tsunehiko Ikeda

  Redox report : communications in free radical research. 01/2010; 15(6):250-8.

  Diabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stressDiabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stress have been identified in the retina. Pericytes are known to be susceptible to oxidative stress and selective dropout of pericytes is one of the earliest pathological changes in DR. Extracellular-superoxide dismutase (EC-SOD) is a major antioxidative enzyme and protects vascular cells from the damaging effects of superoxide. Treatment with own conditioned medium significantly decreased EC-SOD expression in pericytes, while the expression of vascular endothelial growth factor and tumor necrosis factor-α were elevated. The addition of chemical chaperone 4-phenyl butyric acid significantly suppressed the effects of conditioned medium on EC-SOD and GRP78, a prominent ER-resident chaperone. Moreover, the cell viability of pericytes changed in a manner similar to that of EC-SOD expression. These results suggest that the expressions of EC-SOD should be regulated, at least partially, through ER stress. Continuous flow of culture media neutralized the ER-stress triggered decrease of EC-SOD expression. The stagnation of factors related to ER-stress around pericytes might reduce EC-SOD expression under pathophysiological conditions such as retinal edema, and this could induce and/or promote the intraretinal microvascular impairment and development of pathogenesis in DR.

• The effect of hypoxia mimetic cobalt chloride on the expression of EC-SOD in 3T3-L1 adipocytes.

  Authors: Tetsuro Kamiya, Hirokazu Hara, Naoki Inagaki, Tetsuo Adachi

  Redox report : communications in free radical research. 01/2010; 15(3):131-7.

  It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectinIt is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-alpha (TNF-alpha). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl(2)), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-alpha and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-alpha is a key regulator of the reduction of EC-SOD and adiponectin in CoCl(2)-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

• PKC regulation of neuronal zinc signaling mediates survival during preconditioning.

  Authors: Mandar A Aras, Hirokazu Hara, Karen A Hartnett, Karl Kandler, Elias Aizenman

  Journal of neurochemistry. 05/2009;

  Sub-lethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn(2+) liberation mediates neuronal death pathways, we tested whether a sub-lethal increase inSub-lethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn(2+) liberation mediates neuronal death pathways, we tested whether a sub-lethal increase in free Zn(2+) could also trigger neuroprotection. Neuronal free Zn(2+) transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn(2+) that contributed significantly to excitotoxicity in non-preconditioned neurons, but not in tolerant neurons, unless preconditioning-induced free Zn(2+) was chelated. Thus, preconditioning may trigger the expression of Zn(2+)-regulating processes, which, in turn, prevent subsequent Zn(2+)-mediated toxicity. Indeed, preconditioning increased Zn(2+)-regulated gene expression in neurons. Examination of the molecular signaling mechanism leading to this early Zn(2+) signal revealed a critical role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn(2+). We identified a conserved PKC phosphorylation site at serine-32 (S32) of metallothionein (MT) that was important in modulating Zn(2+)-regulated gene expression and conferring excitotoxic tolerance. Importantly, we observed increased PKC-induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn(2+) serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.

• Zinc Induces Expression of the BH3-only Protein PUMA Through p53 and ERK Pathways in SH-SY5Y Neuroblastoma Cells.

  Authors: Hirokazu Hara, Tetsuro Kamiya, Tetsuo Adachi

  Neurochemical research. 03/2009;

  Accumulating evidence suggests that zinc (Zn(2+)) contributes to neuronal death in pathologic states such as ischemia. p53-upregulated modulator of apoptosis (PUMA), which is a BH3-only protein, isAccumulating evidence suggests that zinc (Zn(2+)) contributes to neuronal death in pathologic states such as ischemia. p53-upregulated modulator of apoptosis (PUMA), which is a BH3-only protein, is known to promote apoptosis through a tumor suppressor p53-dependent and -independent mechanism. In this study, we examined the effect of Zn(2+) on the induction of the PUMA gene in human neuroblastoma SH-SY5Y cells. The expression of PUMA was induced by Zn(2+) in a dose- and time-dependent manner. A reporter assay revealed that Zn(2+) activated the PUMA promoter. In addition, the mutation of the p53 binding site in the PUMA promoter region reduced promoter activation by Zn(2+). These findings suggest that p53 participates in Zn(2+)-induced PUMA expression. Furthermore, we also demonstrated here that Zn(2+) stimulates the phosphorylation of ERK and that the MEK-ERK pathway inhibitor, U0126, suppressed Zn(2+)-induced PUMA expression. Taken together, these results indicate that Zn(2+) regulates the induction of PUMA through p53 and ERK pathways.

• An Effort to Improve Advanced Problem-based Learning Tutorial.

  Authors: Tetsuo Adachi, Masumi Suzui, Kuniko Naoi, Tetsuro Kamiya, Hirokazu Hara

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 02/2009; 129(1):177-82.

  To prepare for the introduction of the advanced problem-based learning (PBL) tutorial for higher-grade students under the six-year pharmacy curriculum, a trial of the tutorial was performed in aTo prepare for the introduction of the advanced problem-based learning (PBL) tutorial for higher-grade students under the six-year pharmacy curriculum, a trial of the tutorial was performed in a fourth-grade class under the former four-year curriculum in 2007. A questionnaire survey conducted to identify any problems in performing the tutorial revealed: 1) the number of students in each group was too large; 2) the contents of presentations seemed to overlap due to the limited number of task cases, which forced more than one group to address a particular case; and 3) the time-line from the day of product presentation to that of periodic examination was too short to hold a sufficient group discussion. In 2008, to resolve these problems: 1) the number of groups was increased to reduce the number of students in each group; 2) new task cases were added to decrease the number of groups addressing a particular case; and 3) an adequate time period was arranged between the days of product presentation and periodic examination. The survey conducted this year demonstrated that students' learning environment had been improved by these changes in the method of performing the tutorial, but also revealed new problems such as prolongation of the time required for product presentation and the difficulty levels of task cases. In addition, the usefulness of customer satisfaction (CS) analysis was demonstrated as a result of applying it to the data analysis of evaluation performed on group presentations.

• Expression of extracellular superoxide dismutase during adipose differentiation in 3T3-L1 cells.

  Authors: Tetsuo Adachi, Taisuke Toishi, Haoshu Wu, Tetsuro Kamiya, Hirokazu Hara

  Redox report : communications in free radical research. 02/2009; 14(1):34-40.

  Obesity is known to be the primary causal component in metabolic syndrome. Adipocytes in obese patients exhibit increased oxidative stress via the activation of reactive oxygen speciesObesity is known to be the primary causal component in metabolic syndrome. Adipocytes in obese patients exhibit increased oxidative stress via the activation of reactive oxygen species (ROS)-producing systems and inactivation of antioxidant enzymes. Extracellular superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from the damaging effects of ROS. An earlier report showed that plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to body mass index and homeostasis model assessment-insulin resistance index. Moreover, the administration of pioglitazone, an antidiabetic agent, significantly increased the plasma level of EC-SOD. In this report, the expression of EC-SOD was compared to other adipocytokines in mice 3T3-L1 pre-adipocytes. EC-SOD expression levels were increased after the induction of differentiation and then declined, which was similar to adiponectin and transcription factors such as peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha). On the other hand, the expression levels of pro-inflammatory adipocytokines, such as tumor necrosis factor-alpha (TNF-alpha) and monocyte chemo-attractant protein-1 (MCP-1), increased markedly in the development stage of cells. It was observed that the expression of EC-SOD in differentiated 3T3-L1 cells co-cultured with LPS-stimulated J774 macrophages was up-regulated, while the addition of TNF-alpha down-regulated EC-SOD and adiponectin expression in adipocytes. It is known that infiltrated and activated macrophages produce extracellular ROS at high levels in adipose tissue. It is possible that the expression of EC-SOD in adipocytes was stimulated to protect them from oxidative stress in the co-culture system.

• Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells.

  Authors: Tetsuro Kamiya, Hirokazu Hara, Harutaka Yamada, Hirokazu Imai, Naoki Inagaki, Tetsuo Adachi

  Free radical research. 12/2008;

  It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD)It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl(2))-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl(2).

• Microglia induce neurotoxicity via intraneuronal Zn(2+) release and a K(+) current surge.

  Authors: Megan E Knoch, Karen A Hartnett, Hirokazu Hara, Karl Kandler, Elias Aizenman

  Glia. 02/2008; 56(1):89-96.

  Microglial cells are critical components of the injurious cascade in a large number of neurodegenerative diseases. However, the precise molecular mechanisms by which microglia mediate neuronal cellMicroglial cells are critical components of the injurious cascade in a large number of neurodegenerative diseases. However, the precise molecular mechanisms by which microglia mediate neuronal cell death have not been fully delineated. We report here that reactive species released from activated microglia induce the liberation of Zn(2+) from intracellular stores in cultured cortical neurons, with a subsequent enhancement in neuronal voltage-gated K(+) currents, two events that have been intimately linked to apoptosis. Both the intraneuronal Zn(2+) release and the K(+) current surge could be prevented by the NADPH oxidase inhibitor apocynin, the free radical scavenging mixture of superoxide dismutase and catalase, as well as by 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride. The enhancement of K(+) currents was prevented by neuronal overexpression of metallothionein III or by expression of a dominant negative (DN) vector for the upstream mitogen-activated protein kinase apoptosis signal regulating kinase-1 (ASK-1). Importantly, neurons overexpressing metallothionein-III or transfected with DN vectors for ASK-1 or Kv2.1-encoded K(+) channels were resistant to microglial-induced toxicity. These results establish a direct link between microglial-generated oxygen and nitrogen reactive products and neuronal cell death mediated by intracellular Zn(2+) release and a surge in K(+) currents.

• [Molecular mechanism of neuroprotective drugs against oxidative stress-induced neuronal cell death]

  Authors: Hirokazu Hara

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 09/2007; 127(8):1199-205.

  NF-E2-related factor-2 (Nrf2), a basic leucine zipper transcription factor, is involved in the expression of numerous detoxifying and antioxidant genes via the antioxidant response element (ARE).NF-E2-related factor-2 (Nrf2), a basic leucine zipper transcription factor, is involved in the expression of numerous detoxifying and antioxidant genes via the antioxidant response element (ARE). Keap1, a cytoplasmic protein, sequesters Nrf2 in the cytoplasm under normal conditions. Various stimuli, including electrophiles and oxidative stress, liberate Nrf2 from Keap1, allowing Nrf2 to translocate into the nucleus and to bind to the ARE. Recently, there is increasing evidence that compounds that stimulate the activation of the Nrf2-ARE pathway may become useful therapeutic drugs for neurodegenerative diseases associated with oxidative stress. Apomorphine (Apo), a dopamine D(1)/D(2) receptor agonist, is used for clinical therapy of Parkinson's disease. On the other hand, Apo is a potent radical scavenger and has protective effects on oxidative stress-induced cell death. We previously reported that pretreatment of human neuroblastoma SH-SY5Y cells with Apo enhanced the protective effects. In addition, we have recently demonstrated that Apo stimulates the translocation of Nrf2 into the nucleus and the transactivation of the ARE. Our findings suggest that not only the function as a radical scavenger, but also the function as an Nrf2-ARE pathway activator may be involved in the neuroprotective effects of Apo on oxidative stress-induced neuronal cell death. In this review, our recent studies on the mechanism underlying Apo-induced neuroprotection are summarized.