Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.     

Growth factors prevent changes in Bcl-2 and Bax expression and neuronal apoptosis induced by nitric oxide

Recent studies have shown that nitric oxide (NO) donors can trigger apoptosis of neurons, and growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) can protect against NO-induced neuronal cell death. The purpose of this study was to elucidate the possible mechanisms of NO-mediated neuronal apoptosis and the neuroprotective action of these growth factors. Both IGF-1 and bFGF prevented apoptosis induced by NO donors, sodium nitroprusside (SNP) or 3-morpholinosydnonimin (SIN-1) in hippocampal neuronal cultures. Incubation of neurons with SNP induced caspase-3-like activation following downregulation of Bcl-2 and upregulation of Bax protein levels in cultured neurons. Treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by SNP. In addition, treatment of neurons with an inhibitor of caspase-3, Ac-DEVD-CHO, together with SNP did not affect the changes in the protein levels, although it inhibited NO-induced cell death. Pretreatment of cultures with either IGF-1 or bFGF prior to NO exposure inhibited caspase-3-like activation together with the changes in Bcl-2 and Bax protein levels. These results suggest that the changes in Bcl-2 and Bax protein levels followed by caspase-3-like activation are a component in the cascade of NO-induced neuronal apoptosis, and that the neuroprotective actions of IGF-1 and bFGF might be due to inhibition of the changes in the protein levels of the Bcl-2 family.     

Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1

Cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes. Differentiated chondrocytes in articular cartilage undergo dedifferentiation and apoptosis during arthritis, in which NO production plays a critical role. Here, we investigated the roles and mechanisms of action of insulin-like growth factor-1 (IGF-1) in the chondrogenesis of mesenchymal cells and the maintenance and survival of differentiated articular chondrocytes. IGF-1 induced chondrogenesis of limb bud mesenchymal cells during micromass culture through the activation of phosphatidylinositol 3-kinase (PI3K) and Akt. PI3K activation is required for the activation of protein kinase C (PKC)-alpha and p38 kinase and inhibition of ERK1/2. These events are necessary for chondrogenesis. The growth factor additionally blocked NO-induced dedifferentiation and apoptosis of primary culture articular chondrocytes. NO production in chondrocytes induced down-regulation of PI3K and Akt activities, which was blocked by IGF-1 treatment. Stimulation of PI3K by IGF-1 resulted in blockage of NO-induced activation of p38 kinase and ERK1/2 and inhibition of PKCalpha and PKCzeta, which in turn suppressed dedifferentiation and apoptosis. Our results collectively indicate that IGF-1 regulates differentiation, maintenance of the differentiated phenotype, and apoptosis of articular chondrocytes via a PI3K pathway that modulates ERK, p38 kinase, and PKC signaling.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

Rac1-regulated reactive oxygen species (ROS) production has been implicated in apoptosis. In contrast, pleiotropic protein kinase Akt protects against apoptosis. However, the pro- and antiapoptotic mechanisms of rac1 and Akt, respectively, and the intersection between these mechanisms are incompletely understood. In a model of oxidative stress and apoptosis induced by hypoxia/reoxygenation (H/R) in primary hepatocytes, activation of the PI3-K Akt axis by the prosurvival hepatocyte growth factor (HGF) inhibited H/R-stimulated rac1 activation and intracellular ROS production, and suppressed apoptosis. Suppression of PI3-K or Akt activity abrogated the inhibitory effect of HGF on rac1 activity and rac1-regulated oxidative stress. Furthermore, constitutive activation of Akt or PI3-K in the absence of HGF was sufficient to phosphorylate rac1, inhibit rac1 activation, and suppress rac1-regulated ROS production. These findings demonstrate that growth factor-stimulated activation of PI3-K-Akt is necessary and sufficient to suppress intracellular oxidative stress and apoptosis by inhibiting activation of pro-apoptotic, prooxidative rac1 GTPase.     

Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro

Bone marrow-derived mesenchymal stem cells (MSCs) are of therapeutic interest in a variety of neurological diseases. In this study, we wished to determine whether human MSCs secrete factors which protect cultured rodent cortical neurons from death by trophic factor withdrawal or nitric oxide (NO) exposure. Medium conditioned by MSCs attenuated neuronal death under these conditions, a process which was dependent on intact PI₃kinase/Akt pathway signaling. Trophic withdrawal and NO exposure in cultured cortical neurons led to reduction in Akt signaling pathways, whereas NO administration activated p38 MAPkinase in neuronal cultures. Addition of MSC-conditioned medium significantly activated the PI3kinase/Akt pathway and in neurons exposed to NO, MSC-conditioned medium reduced p38 signaling. We show that MSCs secrete brain-derived neurotrophic factor (BDNF) and addition of anti-BDNF neutralising antibodies to MSC-conditioned medium attenuated its neuroprotective effect. Exposure of neurons to BDNF increased activation of Akt pathways and protected neurons from trophic factor withdrawal. These observations determine the mechanisms of neuroprotection offered by MSC-derived factors and suggest an important role for BDNF in neuronal protection.     

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.     

Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling

Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.     

A predominant apoptotic death pathway of neuronal PC12 cells induced by activated microglia is displaced by a non-apoptotic death pathway following blockage of caspase-3-dependent cascade.

Activated microglia have been implicated in the regulation of neuronal cell death. However, the biochemical mechanism for neuronal death triggered by activated microglia is still unclear. When treated with activated microglia, neuronal PC12 cells undergo apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation. Apoptotic bodies formed were subsequently phagocytosed by neighboring activated microglia. Pretreatment of the cells with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death. Although Bcl-2 overexpression in the cells caused the inhibition of caspase-3-like protease activity and DNA fragmentation and the effective interference of apoptosis induced by deprivation of trophic factors, it could not suppress the activated microglia-induced neuronal death. At the electron microscopic level, degenerating cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular-shaped masses, severely disintegrated perikarya, and marked vacuolation. Various protease inhibitors tested did not inhibit this cell death, whereas the radical oxygen species scavenger N-acetyl-L-cysteine significantly suppressed this death. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade.     

Involvement of transforming growth factor-beta 1 signaling in hypoxia-induced tolerance to glucose starvation

Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and we have reported involvement of AMP-activated protein kinase-alpha (AMPK-alpha), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-alpha phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-alpha expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-alpha activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-beta (TGF-beta) receptor, we showed an involvement of TGF-beta in hypoxia-induced tolerance. TGF-beta1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1alpha-independent manner, and addition of recombinant TGF-beta suppressed cell death during glucose starvation even under normoxic condition. AMPK-alpha, Akt, and ARK5 were activated by TGF-beta1, and Akt and AMPK-alpha phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-beta receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-beta1 through AMPK-alpha activation and the Akt/ARK5 system.