Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation

Reactive oxygen species such as hydrogen peroxide (H₂O₂) are involved in many cellular processes that positively and negatively regulate cell fate. H₂O₂, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H₂O₂ was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H₂O₂-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.     

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.     

Kynurenic acid attenuates MPP(+)-induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinson's disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

PI3K信号通路通过Skp2、p27调节肝癌细胞的增殖

探讨磷脂酰肌醇3-激酶(PI3K)信号通路调节肝癌细胞增殖的机制.用LY294002特异性阻断PI3K信号通路后,人肝癌细胞(SMMC-7721)的增殖明显被抑制.RT-PCR及蛋白质印迹结果显示,LY294002增加了p27蛋白的表达,但不影响p27的mRNA表达.在LY294002处理的细胞中转入p27的RNAi质粒以干扰p27蛋白的表达后,肝癌细胞的增殖能力可部分恢复.放线菌酮(Chx)处理实验表明,阻断PI3K信号通路使p27蛋白的半衰期延长,稳定性增加.进一步研究发现,LY294002可抑制介导p27蛋白降解的关键分子Skp2的mRNA表达,还可缩短Skp2蛋白的半衰期,降低Skp2蛋白的稳定性.但在SMMC-7721中分别转染PI3K下游重要靶分子Akt的持续激活和失活突变体,却并不影响p27蛋白的表达.这些结果表明,PI3K信号通路在转录及翻译后水平调节Skp2的表达而影响p27蛋白的降解,从而调节肝癌细胞的增殖,但Akt并没有参与这种调节.     

Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons

In the present study we focused in the PI3K/Akt pathway which plays a key role in neuronal survival. Here we show that inhibition of PI3K/Akt by means of LY294002 induces apoptosis via a caspase-dependent and calpain-independent pathway in cerebellar granule neurons (CGNs). This finding was confirmed using zVAD-fmk, a widely caspase inhibitor that prevents apoptosis. For this purpose, we compared two models of apoptosis in CGNs, namely inhibition of PI3K/Akt, and serum potassium deprivation (S/K deprivation). In contrast to the S/K deprivation model, caspase-3 was not activated when PI3K is inhibited. Likewise, CDK5 activation was not involved in this apoptotic process, because calpain activation is responsible for the formation of CDK5/p25 neurotoxic form. However, S/K deprivation activated calpain, as it is shown by α-spectrin breakdown, and favoured the formation of CDK5/p25. Moreover, although PI3K/Akt inhibition enhanced pRbser780 phosphorylation, no increase in the expression of cell-cycle proteins, namely: cyclin D, cyclin E, CDK2 or CDK4, was detected. Furthermore, BrdU incorporation assay did not shown any increase in DNA synthesis. Likewise, PI3K/Akt inhibition increased GSK3β activity and c-Jun phosphorylation, which implicates these two pathways in this apoptotic route. Although previous reports suggest that apoptosis induced in CGNs by LY294002 and S/K deprivation causes PI3K inhibition and increases GSK3β activity and c-Jun phosphorylation activation, our results demonstrate substantial differences between them and point to a key role of GSK3β in the apoptosis induced in CGNs in the two models tested.     

Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.     

The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3β and ERK1/2 signaling pathway

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.     

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.     

Erythropoietin prevents PC12 cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis via the Akt/GSK-3β/caspase-3 mediated signaling pathway

Apoptosis is a contributing cause of dopaminergic neuron loss in Parkinson disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that exposure of PC12 cells to 1-methyl-4-phenylpyridinium ion (MPP⁺) with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL and caspase-3 activity when compared to MPP⁺ treatment alone. EPO induced sustained phosphorylation of Akt and its substrate, GSK-3β, reduced caspase-3 activities in PC12 cells. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, the specific blocker of phosphatidylinositol 3-kinase (PI3K). The effects of EPO on GSK-3β and caspase-3 activities were also blocked by LY294002. LiCl, the inhibitor of GSK-3β, downregulated the caspase-3 activity and blocked the apoptosis induced by MPP⁺. Finally, we determined that EPO transiently activated the ERK signaling pathway, but PD98059, a specific inhibitor of ERK, does not alter the survival effect of EPO in this model system. Thus, these findings indicate that EPO protects against apoptosis in PC12 cells exposed to MPP⁺, through the Akt/GSK-3β/caspase-3 signaling pathway, but the ERK pathway is not involved in the EPO-dependent survival enhancing effect in this model system. The authors Yan Wu and You Shang are equally contributed to this work.     

Overexpression of interferon-gamma-inducible GTPase inhibits coxsackievirus B3-induced apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway and inhibition of viral replication

Our previous studies using differential mRNA display have shown that interferon-gamma-inducible GTPase (IGTP), was up-regulated in coxsackievirus B3 (CVB3)-infected mouse hearts. In order to explore the effect of IGTP expression on CVB3-induced pathogenesis, we have established a doxycycline-inducible Tet-On HeLa cell line overexpressing IGTP and have analyzed activation of several signaling molecules that are involved in cell survival and death pathways. We found that following IGTP overexpression, protein kinase B/Akt was strongly activated through phosphorylation, which leads to phosphorylation of glycogen synthase kinase-3 (GSK-3). Furthermore, in the presence of CVB3 infection, the intensity of the phosphorylation of Akt was further enhanced and associated with a delayed activation of caspase-9 and caspase-3. These data indicate that IGTP expression appears to confer cell survival in CVB3-infected cells, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell viability assay. However, the ability of IGTP to induce phosphorylation of Akt and to promote cell survival was attenuated by the phosphotidylinositol-3 kinase (PI3-K) inhibitor LY294002. Transient transfection of the cells with a dominant negative Akt construct followed by doxycycline induction and CVB3 infection reversed Akt phosphorylation to basal levels and returned caspase-3 activity to levels similar to those when the PI3-K inhibitor LY294002 was added. Moreover, IGTP expression inhibited viral replication and delayed CVB3-induced cleavage of eukaryotic translation initiation factor 4G, indicating that IGTP-mediated cell survival relies on not only the activation of PI3-K/Akt, inactivation of GSK-3 and suppression of caspase-9 and caspase-3 but also the inhibition of viral replication.     

Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates autophagy and induces apoptosis through p53 pathway in gastric cancer cell line SGC7901

We aimed to study the effects of LY294002, an inhibitor of class I phosphatidylinositol 3-kinase (PI3K), on proliferation, apoptosis, and autophagy in gastric cancer cell line SGC7901. In this study, we showed that LY294002 inhibited the viability of gastric cancer SGC7901 cells. We also showed that LY294002 increased the expression of microtubule-associated protein 1 light chain 3 (LC3), and increased monodansylcadaverine (MDC)-labeled vesicles. LY294002 activated autophagy by activating p53 and caspase-3, and induced apoptosis by up-regulatingp53 and p53-up-regulated modulator of apoptosis ( PUMA ). Therefore, LY294002 might induce cytotoxicity in SGC7901 cells through activation of p53 and the downstream point PUMA . These findings suggest that inhibition of the class I PI3K signaling pathway is a potential strategy for managing gastric cancers.     

Inhibition of the PI3K pathway sensitizes fludarabine-induced apoptosis in human leukemic cells through an inactivation of MAPK-dependent pathway

In the present study, we have investigated the effects of PI3K/Akt pathway on the response of human leukemia cells to fludarabine. Inhibition of PI3K/Akt pathway with a selective inhibitor (e.g., LY294002, or wortmannin) in leukemic cells markedly potentiated fludarabine-induced apoptosis. Inhibition of the PI3K/Akt downstream target mTOR by rapamycin also significantly enhanced fludarabine-induced apoptosis. The co-treatment of fludarabine/LY294002 resulted in significant attenuation in the levels of both phospho-Erk1/2 and phospho-Akt, as well as a marked increase in the level of phospho-JNK. The broad spectrum caspase inhibitor BOC-D-fmk markedly blocked fludarabine/LY-induced apoptosis, had no effect on cytochrome c release to the cytosol, and did abrogate caspase and PARP cleavage. This indicates that mitochondrial dysfunction is upstream of the caspase cascade. Moreover, constitutive activation of the MEK/Erk pathway completely blocked apoptosis induced by the combination of fludarabine/LY294002. Additionally, either constitutive activation of Akt or blockage of the JNK pathway significantly diminished apoptosis induced by the combination. Collectively, these findings demonstrate that inactivation of MAPK, Akt, and activation of the JNK pathway contributes to the induction of apoptosis induced by fludarabine/LY. Comparatively, MAPK inactivation plays a crucial role in fludarabine/LY-induced apoptosis. These results also strongly suggest that combining fludarabine with an inhibitor of the PI3K/Akt/mTOR pathway may represent a novel therapeutic strategy for hematological malignancies.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

PI3K pathway inhibitor LY294002 alters Jurkat T cell biobehaviours via ERK1/2-ICBP90 mediation

Little is known about whether there is a relationshipbetweenPI3K/AKT, ERK1/2 and an inverted CCAAT box binding protein (ICBP90) in biological behaviours of tumour cells. The aim of this study was to determine thisusing Jurkat T cells. Compared to PD98059 (an ERK1/2 signaling inhibitor), DAPT (a Notch signaling inhibitor) or adriamycin (a classical anti-tumour drug), the inhibition of Jurkat T cell growth by LY294002 (a PI3K/Akt signaling inhibitor) was more obvious. LY294002 combined with adriamycin appeared to have a synergistic effect. LY294002 strongly blocked Jurkat T cells at each phase of cell cycle with a decrease of DNA content, superior to adriamycin. Consistent with these changes, the expression of phosphorylated ERK1/2 was markedly decreased in the LY294002-treated Jurkat T cells, leading to the reduction of ICBP90 production, followed by moderate attenuation of TGF-β secretion. The results suggest that deactivation of PI3K/Akt signalling can surpress Jurkat T cell growth through inhibiting cell proliferation and blocking the cell cycle. ICBP90 may mediate the PI3K/AKT-ERK1/2 signalling to regulate leukemia cell growth.     

Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase

Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling promotes expansion of the mucosal epithelium indirectly via activation of growth and anti-apoptotic pathways; however, the cellular mechanisms coupling direct GLP-2R activation to cell survival remain poorly understood. We now demonstrate that GLP-2, in a cycloheximide-insensitive manner, enhanced survival in baby hamster kidney cells stably transfected with the rat GLP-2R; reduced mitochondrial cytochrome c efflux; and attenuated the caspase-dependent cleavage of Akt, poly(ADP-ribose) polymerase, and beta-catenin following inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002. The prosurvival effects of GLP-2 on LY294002-induced cell death were independent of Akt, p90(Rsk), or p70 S6 kinase activation; were mimicked by forskolin; and were abrogated by inhibition of protein kinase A (PKA) activity. GLP-2 inhibited activation of glycogen synthase kinase-3 (GSK-3) through phosphorylation at Ser(21) in GSK-3alpha and at Ser(9) in GSK-3beta in a PI3K-independent, PKA-dependent manner. GLP-2 reduced LY294002-induced mitochondrial association of endogenous Bad and Bax and stimulated phosphorylation of a transfected Bad fusion protein at Ser(155) in a PI3K-independent, but H89-sensitive manner, a modification known to suppress Bad pro-apoptotic activity. These results suggest that GLP-2R signaling enhances cell survival independently of PI3K/Akt by inhibiting the activity of a subset of pro-apoptotic downstream targets of Akt in a PKA-dependent manner.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Inhibition of Src-like kinases reveals Akt-dependent and -independent pathways in insulin-like growth factor I-mediated oligodendrocyte progenitor survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.     

The Survival of Sympathetic Neurons Promoted by Potassium Depolarization, but Not by Cyclic AMP, Requires Phosphatidylinositol 3-Kinase and Akt

Phosphatidylinositol (PI) 3-kinase and Akt protein kinase mediate trophic factor-dependent survival in certain neurons. However, a role for these enzymes in neuronal survival promoted by other agents is unclear. We have tested PI 3-kinase and Akt for their role in survival promoted by membrane-depolarizing concentrations of extracellular potassium and the cell-permeable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (cpt-cAMP). Depolarization of sympathetic neurons resulted in an increase in the activities of both PI 3-kinase and Akt. In addition, the PI 3-kinase inhibitor LY294002 was a potent inducer of cell death in depolarized neurons. Stimulation with cpt-cAMP resulted in relatively small increases in PI 3-kinase and Akt activities, and neurons maintained with cpt-cAMP were more resistant to LY294002-induced death than were depolarized neurons. Expression of either dominant-negative PI 3-kinase or dominant-negative Akt blocked survival promoted by depolarization but not by cpt-cAMP. These results indicate that a PI 3-kinase/Akt pathway is required for survival of sympathetic neurons mediated by depolarization but not by cpt-cAMP. Thus, the survival of sympathetic neurons can be maintained through PI 3-kinase/Akt-dependent and -independent pathways.