Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.     

Kainic acid-induced apoptosis in rat striatum is associated with nuclear factor-kappaB activation

The present study evaluated whether nuclear factor-kappaB (NF-kappaB) activation contributes to the apoptotic-like death of striatal neurons induced by kainic acid (KA) receptor stimulation. Intrastriatally infused KA (1.25-5.0 nmol) produced substantial neuronal loss as indicated by an 8-73% decrease in 67-kDa glutamic acid decarboxylase (p<0.05). KA (1.25-5.0 nmol) elicited internucleosomal DNA fragmentation that was inhibited by the AMPA/KA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dibenzo[f]quinoxaline-7-sulfonamide) but not by the NMDA receptor antagonist MK-801. A decrease in IkappaB-alpha protein levels, which was accompanied by an increase in NF-kappaB binding activity, was found from 6 to 72 h after KA (2.5 nmol) infusion. NF-kappaB was composed mainly of p65 and c-Rel as revealed by supershift assay. In addition, c-Myc and p53 increased from five- to sevenfold from 24 to 72 h after KA (2.5 nmol) administration. Immunohistochemistry revealed high levels of c-Myc and p53 immunoreactivity, mainly in medium-sized striatal neurons. Pretreatment with the cell-permeable recombinant peptide NF-kappaB SN50 (5-20 microg) blocked NF-kappaB nuclear translocation, but had no effect on AP-1 binding. NF-kappaB SN50 also inhibited the KA-induced up-regulation of c-Myc and p53, as well as internucleosomal DNA fragmentation. The apoptotic-like destruction of rat striatal neurons induced by KA receptor stimulation thus appears to involve biochemical mechanisms similar to those mediating the excitotoxic response to NMDA receptor stimulation. The present results provide additional support for the view that NF-kappaB activation contributes to c-Myc and p53 induction and subsequent apoptosis in an excitotoxic model of Huntington's disease.     

Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.     

Regulation of tumor necrosis factor-α expression by CD40 ligation in BV-2 microglial cells

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor-alpha (TNF-alpha) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF-alpha in BV-2 microglial cells. There was very little induction of TNF-alpha by ligation of CD40 alone by either cross-linking antibodies against CD40 or recombinant CD40 ligand (CD154). The absence of any increase in TNF-alpha production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF-alpha. However, CD40 ligation markedly induced the production of TNF-alpha as well as the expression of TNF-alpha mRNA in interferon-gamma (IFN-gamma)-stimulated BV-2 glial cells. Ligation of CD40 in CD40-overexpressed cells markedly enhanced the expression of TNF-alpha in the presence of IFN-gamma. To understand the mechanism of CD40 ligation-mediated induction/stimulation of TNF-alpha, we investigated the role of nuclear factor-kappaB (NF-kappaB) and C/EBPbeta. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF-kappaB and not that of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of TNF-alpha and that the activation of C/EBPbeta is also necessary for strong induction of TNF-alpha. Consistently, a dominant-negative mutant of p65 (Delta(p65)) and that of C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of TNF-alpha in BV-2 microglial cells stimulated with the combination of IFN-gamma and CD40 ligand. Taken together, these studies suggest that activation of both NF-kappaB and C/EBPbeta is important for strong induction of TNF-alpha and that CD40 ligation regulates the expression of TNF-alpha by modulating the activation of only NF-kappaB but not that of C/EBPbeta.     

Lovastatin inhibits the growth and survival pathway of phosphoinositide 3-kinase/protein kinase B in immortalized rat brain neuroblasts

We previously showed that lovastatin, an HMG-CoA reductase inhibitor, suppresses cell growth by inducing apoptosis in rat brain neuroblasts. Our aim was to study intracellular signalling induced by lovastatin in neuroblasts. Lovastatin significantly decreases the phosphoinositide 3-kinase (PI3-K) activity in a concentration-dependent manner. Expression of p85 subunit and its association with phosphotyrosine-containing proteins are unaffected by lovastatin. Lovastatin decreases protein kinase B (PKB)/Akt phosphorylation, and its downstream effectors, p70^S6K and the eukaryotic initiation factor 4E (eIF4E) regulatory protein 1, 4E-BP1, in a concentration-dependent manner, and reduces p70^S6K expression. Lovastatin effects are fully prevented with mevalonate. Only the highest dose of PI3-K inhibitors that significantly reduce PI3-K kinase activity induces apoptosis in neuroblasts but to a lower degree than lovastatin. In summary, this work shows that treatment of brain neuroblasts with lovastatin leads to an inhibition of the main pathway that controls cell growth and survival, PI3-K/PKB and the subsequent blockade of downstream proteins implicated in the regulation of protein synthesis. This work suggests that inactivation of the antiapoptotic PI3-K appears insufficient to induce the degree of neuroblasts apoptosis provoked by lovastatin, which must necessarily involve other intracellular pathways. These findings might contribute to elucidate the molecular mechanisms of some statins effects in the central nervous system.     

Effect of the Alzheimer amyloid fragment Abeta(25-35) on Akt/PKB kinase and survival of PC12 cells

The phosphatidylinositol 3 kinase (PI3K)-Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Abeta), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Abeta-derived Abeta(25-35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Abeta(25-35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Abeta(25-35) and Abeta(1-40) with a kinetics different to that of nerve-derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Abeta(25-35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Abeta(25-35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane-targeted N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP-transfected cells. The present findings demonstrate that Akt1 is activated in response to Abeta(25-35) in a PI3K-dependent manner and that active Akt1 protects PC12 cells against the pro-apoptotic action of this peptide.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Leumorphin has an anti-apoptotic effect by activating epidermal growth factor receptor kinase in rat pheochromocytoma PC12 cells

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, beta-neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor-binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition-binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src-dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation-induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, which occur independent of the kappa opioid receptor.     

Activation of nuclear transcription factor kappa B (NF-kappaB) is essential for dopamine-induced apoptosis in PC12 cells

The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF-kappaB) is activated by oxidative stress, we studied the involvement of NF-kappaB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1-0.5 m M) treatment increased the phosphorylation of the IkappaB protein, the inhibitory subunit of NF-kappaB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF-kappaB-p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine-induced NF-kappaB activation was also evidenced by electromobility shift assay using radioactive labeled NF-kappaB consensus DNA sequence. Cell-permeable NF-kappaB inhibitor SN-50 rescued the cells from dopamine-induced apoptosis and showed the importance of NF-kappaB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF-kappaB-p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF-kappaB activation is essential to dopamine-induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.     

Matriptase is required for the active form of hepatocyte growth factor induced Met,focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.     

Concomitant induction of apoptosis and expression of monocyte chemoattractant protein-1 in cultured rat luteal cells by nuclear factor-kappaB and oxidative stress

It has previously been shown that expression of monocyte chemoattractant protein (mcp)-1 and apoptosis of luteal cells occur concomitantly during the estrous cycle in the rat corpus luteum; however, luteal cells containing mcp-1 mRNA did not seem to be apoptotic. In the present study, the relationship between the induction of apoptosis and mcp-1 expression in cultures of dispersed rat luteal cells was examined. Both apoptosis and mcp-1 expression were spontaneously induced in cultured luteal cells in a manner inhibitable by antioxidative reagents or an inhibitor of nuclear translocation of nuclear factor-kB. However, the cells containing mcp-1 mRNA were distinct from those undergoing apoptosis, and the inhibition of apoptosis by the pan-caspase inhibitor z-VAD-fmk did not influence the induction of mcp-1 expression. These results collectively indicate that oxidative stress simultaneously, but independently, induces apoptosis and mcp-1 expression in luteal cells through the activation of nuclear factor-kB. This phenomenon might help to explain how monocytes/macrophages accumulate in regressive corpora lutea where their target apoptotic cells exist.     

PEMT2过表达抑制大鼠肝癌细胞PI3K, Akt的表达(英)

为探讨磷脂酰乙醇胺-N-甲基转移酶2(PEMT2)过表达抑制大鼠肝癌细胞增殖的机制,构建了PEMT2高表达细胞克隆,并采用半定量RT-PCR、免疫细胞化学及流式细胞仪技术,研究了PEMT2过表达对PI3K/Akt信号转导途径的影响.实验结果显示,PEMT2过表达可抑制细胞PI3K和Akt的表达,并诱导细胞凋亡.这一结果提示,PI3K/Akt信号转导途径下调可能是PEMT2抑制肝癌细胞增殖的部分机制.     

Aplasia Ras homologue member I overexpression induces apoptosis through inhibition of survival pathways in human hepatocellular carcinoma cells in culture and in xenograft

The aim of the present study was to determine the effects of ARHI (aplasia Ras homologue member I; also known as DIRAS3), a member of the Ras superfamily, on HCC (hepatocellular carcinoma) cells and to define the molecular pathways involved. Stable transfection of ARHI into the HCC cell line Hep3B that lacks expression of this gene reduced cell proliferation significantly as compared with the transfection of empty vector (P<0.01). Moreover, the re-expression of ARHI induced significant apoptosis, whereas a few vector transfectants or non-transfected cells displayed apoptosis. Mechanistically, ARHI restoration impeded the activation of both Akt (also called protein kinase B) and NF-κB (nuclear factor κB). In vivo, restoring ARHI also exerted suppressive effects on xenograft tumour growth, which was coupled with increased apoptosis. Together, these results indicate that ARHI has pro-apoptotic effects on HCC cells, which is associated with the inactivation of both Akt and NF-κB survival pathways.