NMDA neuroprotection against a phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of glycogen synthase kinase-3beta-induced apoptosis

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.     

Decreased apoptosis in polyamine depleted IEC-6 cells depends on Akt-mediated NF-κB activation but not GSK3β activity

The PI3-kinase/Akt pathway promotes cell survival in many different cell types including intestinal epithelial cells. Increased AKT activation in polyamine depleted intestinal epithelial cells correlated well with the decrease in TNF-α-induced apoptosis. Increased Akt activation and GSK3β (Ser 9) phosphorylation without significant effect on Bad (Ser136) phosphorylation indicate that Akt-mediated protection is independent of Bad phosphorylation but may depend on GSK3β. Pretreatment of polyamine-depleted cells with LY294002 increased caspase-9 and caspase-3 activation and decreased basal levels of GSK-3β phosphorylation. Inhibition of GSK3β activity using AR-A014418 or lithium chloride or siRNA-mediated downregulation of its expression had no effect on apoptosis. Inhibition of PI3-kinase and over-expression of dominant negative Akt (DN-AKT), significantly increased apoptosis in polyamine depleted cells. DN-Akt expression reversed the protective effect of polyamine depletion on apoptosis. DN-Akt, as well as the PI3-kinase inhibitors, prevented Akt activation and subsequent translocation of NF-κB to the nucleus. Constitutively active Akt (CA-AKT) expression increased resistance to TNF-α-induced apoptosis. Constitutively active-Akt expression increased nuclear staining of NF-κB. Moreover, polyamine depletion of DN-Akt cells prevented basal and TNF-α-induced IκBα phosphorylation. Prevention of NF-κB activation in DN-IκBα-transfected cells increased apoptosis in control cells and restored it in polyamine-depleted cells to control levels. These data indicate that Akt regulates the mitochondrial pathway, preventing activation of caspase-9 and thereby caspase-3 via NF-κB and these effects are independent of GSK-3β activity.     

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.     

The Akt/GSK-3beta axis as a new signaling pathway of the histamine H(3) receptor

Drugs targeting the histamine H(3) receptor (H(3)R) are suggested to be beneficial for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. The H(3)R activates G(i/o)-proteins to inhibit adenylyl cyclase activity and modulates phospholipase A(2) and MAPK activity. Herein we show that, in transfected SK-N-MC cells, the H(3)R modulates the activity of the Akt/Glycogen synthase kinase 3beta (GSK-3beta) axis both in a constitutive and agonist-dependent fashion. H(3)R stimulation with the H(3)R agonist immepip induces the phosphorylation of both Ser473 and Thr308 on Akt, a serine/threonine kinase that is important for neuronal development and function. The H(3)R-mediated activation of Akt can be inhibited by the H(3)R inverse agonist thioperamide, and by Wortmannin, LY294002 and PTX, suggesting the observed Akt activation occurs via a G(i/o)-mediated activation of phosphoinositide-3-kinase. H(3)R activation also results in the phosphorylation of Ser9 on GSK-3beta, which acts downstream of Akt and has a prominent role in brain function. In addition, we show the H(3)R-mediated phosphorylation of Akt at Ser473 to occur in primary rat cortical neurons and in rat brain slices. The discovery of this signaling property of the H(3)R adds new understanding to the roles of histamine and the H(3)R in brain function and pathology.     

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.     

Phosphatidylinositol 3-kinase/Akt auto-regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts

It has been reported that platelet-derived growth factor (PDGF)-BB stimulates the synthesis of interleukin (IL)-6 in osteoblasts. In the present study, we investigated whether the phosphatidylinositol 3-kinase (PI3K)/Akt is involved in the PDGF-BB-induced IL-6 synthesis in osteoblast-like MC3T3-E1 cells. PDGF-BB markedly induced the phosphorylation of Akt and GSK-3beta. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, significantly amplified the synthesis of IL-6 by PDGF-BB. The PDGF-BB-induced GSK-3beta phosphorylation was suppressed by the Akt inhibitor. The IL-6 synthesis stimulated by PDGF-BB was markedly enhanced by LY294002 and wortmannin, inhibitors of PI3K. Wortmannin and LY294002 suppressed the PDGF-BB-induced phosphorylation of Akt and GSK-3beta. Taken together, these results strongly suggest that PI3K/Akt negatively regulates the PDGF-BB-stimulated IL-6 synthesis in osteoblasts.     

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.     

AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.     

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear whether GLP-2 can directly stimulate protein synthesis. The objective was to test critically whether GLP-2 receptor (GLP-2R) activation directly stimulates protein synthesis through a PI 3-kinase-dependent Akt-mTOR signaling pathway. HEK 293 cells (transfected with human GLP-2R cDNA) were treated with human GLP-2 with/without pretreatment of PI 3-kinase inhibitor (LY-294002) or mTOR inhibitor (rapamycin). Results show that 1) GLP-2 specifically bound to GLP-2R overexpressed in the HEK cells with K(a) = 0.22 nM and B(max) = 321 fmol/μg protein; 2) GLP-2-stimulated protein synthesis was dependent on the amount of GLP-2R cDNA and the dosage of GLP-2 and reached the plateau among 0.2-2 nM GLP-2; 3) GLP-2-stimulated protein synthesis was abolished by the PI 3-kinase inhibitor and mTOR inhibitor; and 4) GLP-2-mediated stimulation of phosphorylation on Akt and mTOR was dependent on the amount of GLP-2R cDNA transfected and the dosage of GLP-2. In addition, GLP-2-mediated action and signaling in regulation of protein synthesis were confirmed in mouse hippocampal neurons (expressing native GLP-2R). GLP-2 directly stimulated protein synthesis of primary cultured neurons in dosage-dependent, PI 3-kinase-dependent, and rapamycin-sensitive manners, which linked with activation of Akt-mTOR signaling pathway as well. We conclude that GLP-2R activation directly stimulates protein synthesis by activating the PI 3-kinase-dependent Akt-mTOR signaling pathway. GLP-2-stimulated protein synthesis may be physiologically relevant to maintaining neuronal long-term potentiation and providing secondary mediators (namely neuropeptides or growth factors).     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.     

Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: Involvement of PI3K/Akt and GSK-3 beta pathway

Erythropoietin (EPO) prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the neuroprotective effect of EPO in ketamine-induced neurotoxicity in primary cortical neurons. EPO in combination with ketamine greatly increased the cell viability and reduced the number of TUNEL-positive cells. To elucidate a possible mechanism by which EPO exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase pathway using LY294002. The neuroprotection of EPO was prevented by LY294002. Immunoblotting revealed that EPO induced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3β). Moreover, the caspase-3-like activity was increased by addition of ketamine, and decreased by administration of ketamine with EPO. Decreased caspase-3-like activity by administration of ketamine with EPO was restored by LY294002. Our results suggest that PI3K/Akt and GSK-3β pathway are involved in the neuroprotective effect of EPO. You Shang and Yan Wu have contributed equally to this work.     

Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3beta pathway

The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT, which is involved in cell survival, proliferation, and growth, has become a major focus in targeting cancer therapeutics. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was previously identified as a gene induced by several anti-tumorigenic compounds including nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor gamma ligands, and dietary compounds. NAG-1 has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro. In this report, we showed a PI3K/AKT/glycogen synthase kinase-3beta (GSK-3beta) pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3beta. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1l-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor) also induced NAG-1 expression. LY294002 caused increased apoptosis, cell cycle, and cell growth arrest in HCT-116 cells. Inhibition of GSK-3beta, which is negatively regulated by AKT, using AR-A014418 and lithium chloride completely abolished LY294002-induced NAG-1 expression as well as the NAG-1 promoter activity. Furthermore, the down-regulation of GSK-3 gene using small interference RNA resulted in a decline of the NAG-1 expression in the presence of LY294002. These data suggest that expression of NAG-1 is regulated by PI3K/AKT/GSK-3beta pathway in HCT-116 cells and may provide a further understanding of the important role of PI3K/AKT/GSK-3beta pathway in tumorigenesis.     

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.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3

Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.     

Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3β/β-catenin signaling pathway

Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated Akt(Ser473) and GSK-3β(Ser9) than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.