Toshihiko Ikeuchi

THE NEUROTROPHIC ACTION AND SIGNALLING OF EPIDERMAL GROWTH FACTOR

Epidermal growth factor (EGF) is a conventional mitogenic factor that stimulates the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signalling and subsequent effects. The EGFR is expressed in neurons of the cerebral cortex, cerebellum, and hippocampus in addition to other regions of the central nervous system (CNS). In addition, EGF is also expressed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic neurons. In fact, many studies have indicated that EGF has neurotrophic or neuromodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival. On the other hand, EGF also acts on other cell types, including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells. Evidence of the effects of EGF on neurons in the CNS is accumulating, but the mechanisms of action remain essentially unknown. EGF-induced signalling in mitotic cells is better understood than that in postmitotic neurons. Studies of cloned pheochromocytoma PC12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The sustained intracellular signalling correlates with the decreased rate of EGFR down-regulation, which might determine the response of neuronal cells to EGF. It is likely that EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postmitotic neurons.

Insulin Receptor Substrate (IRS)-1 and IRS-2 Are Tyrosine-phosphorylated and Associated with Phosphatidylinositol 3-Kinase in Response to Brain-derived Neurotrophic Factor in Cultured Cerebral Cortical Neurons

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival of various types of neurons in the central nervous system. BDNF binds to and activates the tyrosine kinase receptor, TrkB, initiating intracellular signaling and exerting its effects. Phosphatidylinositol 3-kinase (PI3-K), which has been implicated in promotion of neuronal survival by neurotrophic factors, is a component in the signaling pathway of BDNF. We examined how BDNF activates PI3-K in cultured cerebral cortical neurons. We found that insulin receptor substrate (IRS)-1 and -2 are involved in the BDNF signaling pathway that activates PI3-K. IRS-1 and -2 were tyrosine-phosphorylated and bound to PI3-K in response to BDNF. This BDNF-stimulated signaling via IRS-1 and -2 was inhibited by K-252a, an inhibitor of Trk tyrosine kinase. In addition, signaling via IRS-1 and -2 was markedly sustained as well as the BDNF-induced tyrosine phosphorylation of TrkB. On the other hand, we observed no association of PI3-K with TrkB in response to BDNF. These results indicate that the activation of TrkB by BDNF induces the activation of PI3-K via IRS-1 and -2 rather than by a direct interaction of TrkB with PI3-K in cultured cortical neurons.

Nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1u2003µm. The maximal level of nicotine‐induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)‐induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) α7 subunit‐selective inhibitors had no significant effect on nicotine‐induced ERK phosphorylation. l‐Type voltage‐sensitive calcium channel antagonists inhibited nicotine‐induced ERK phosphorylation. Calcium imaging experiments showed that α7‐containing nAChR subtypes were functional at 1u2003µm of nicotine in the nicotine‐induced calcium influx, and non‐α7 nAChRs were prominent in the Ca2+ influx at 50u2003µm of nicotine. An expression of dominant inhibitory Ras inhibited nicotine‐induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine‐induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non‐α7 nAChRs are involved in nicotine‐induced ERK phosphorylation through CaM kinase and the Ras‐MAP kinase cascade and most of the nicotine‐induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.

Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells.

Abstract The compound 13- l -hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD 50 =about 8 μ M). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N′ -diphenyl- p -phenylenediamine (DPPD), vitamin E and N -acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca 2+ -specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 μ M, did not increase the intracellular Ca 2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca 2+ .

Differences in survival‐promoting effects and intracellular signaling properties of BDNF and IGF‐1 in cultured cerebral cortical neurons

Brain‐derived neurotrophic factor (BDNF) and insulin‐like growth factor‐1 (IGF‐1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival‐promoting effects of BDNF and IGF‐1 on serum deprivation‐induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF‐1 in the neurons. We found that the survival‐promoting effect of BDNF was much weaker than that of IGF‐1 in serum deprivation‐induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3‐kinase (PtdIns3‐K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF‐1 in the cultured cortical neurons, although many reports suggest that PtdIns3‐K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen‐activated protein kinase (MAPK) and cAMP responsive element‐binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF‐1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3‐K‐Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival‐promoting effects of BDNF and IGF‐1.

Generation of reactive oxygen species, release of L-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture.

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O2- and hydrogen peroxide (H2O2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O2-, H2O2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O2- and H2O2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of L-glutamate and activation of caspases are activated during the death induced by high oxygen.

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons.

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mmpotassium (high K+), changing to medium containing 5 mm potassium (low K+; LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells.In vitro kinase assay using glutathioneS-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.

Inhibition of phosphatidylinositol 3-kinase activity elevates c-Jun N-terminal kinase activity in apoptosis of cultured cerebellar granule neurons.

Abstract Cerebellar granule neurons maintained in medium containing 26 mM potassium or in medium (5 mM potassium) with 50 ng/ml brain-derived neurotrophic factor (BDNF) undergo an apoptotic cell death when exposed to 10 μM LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). To investigate the intracellular signaling mechanism of LY294002-induced apoptosis, the activities of Akt and c-Jun N-terminal kinase (JNK) were measured in cells in HK (26 mM potassium) medium or LK+ (5 mM potassium) medium containing BDNF, with or without 10 μM LY294002. Akt activity decreased following the addition of 10 μM LY294002. In addition, we found that LY294002 increased the JNK activity, which is known to mediate some types of cell death in cultured PNS neurons. We also observed elevated expression of c-Jun by LY294002 in HK+ or LK++BDNF. These findings demonstrated that apoptosis induced by inhibition of PI3-K activity involves suppression of the Akt activity and elevation of the JNK activity in cultured cerebellar granule neurons. Our results suggested that the PI3-K-Akt pathway suppresses the activation of JNK and c-Jun expression, and as a result prevents the neuronal cell death in cerebellar granule neurons.

Involvement of phosphatidylinositol-3 kinase in prevention of low K+-induced apoptosis of cerebellar granule neurons

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis.

The molecular mechanism of Bcl-2 phosphorylation and its relationship to Bax is largely unknown. Here we show that the phosphorylation of Bcl-2 is involved in the intracellular translocation of Bax from cytosol to mitochondria in NO-induced neuronal apoptosis. We examined how the phosphorylation of Bcl-2 is regulated during the apoptosis and found it to be mediated by the activation of p38 and ERK, members of the MAPK superfamily. Furthermore, we investigated whether Bcl-2 phosphorylation affected Bax translocation, using mutant Bcl-2 expression vectors. Cortical neuronal cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) prevented the translocation of Bax. In contrast, transfection with Bcl-2 (S70D), a constitutively active Bcl-2 mutant, enhanced the translocation. Our results suggested that Bcl-2 phosphorylated at Ser-70 plays a critial role in the translocation of Bax from the cytosol to the mitochondria, and this may regulate NO-induced neuronal apoptosis.