Activation of extracellular signal-regulated kinase couples platelet-activating factor-induced adhesion and delayed apoptosis of human neutrophils

Platelet-activating factor (PAF) promotes adhesion of neutrophil granulocytes to the endothelium, which is also linked to neutrophil survival. Here we report that PAF can prolong neutrophil survival by suppressing spontaneous apoptosis. PAF induced concurrent activation of the Ras/Raf-1/mitogen-activated protein kinase kinase (MAPKK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt pathways. ERK activation tightly correlated with up-regulation of CD11b/CD18 expression and beta(2)-integrin-dependent homotypic adhesion. These actions of PAF were markedly attenuated by the MAPKK/ERK inhibitor PD98059, but not by the phosphatidylinositol 3-kinase inhibitor wortmannin. By contrast, concurrent activation of ERK and Akt was required to inhibit caspase-3 activation and consequently to delay apoptosis. Consistently, pharmacological inhibition of either ERK or Akt partially reversed the anti-apoptotic action of PAF; however, they did not produce additive inhibition. These results indicate that PAF-induced activation of ERK contributes to both the expression of the pro-adhesive phenotype and repression of neutrophil apoptosis, thereby amplifying the inflammatory response.     

Molecular mechanisms contributing to glutamine-mediated intestinal cell survival

Glutamine, the most abundant amino acid in the bloodstream, is the preferred fuel source for enterocytes and plays a vital role in the maintenance of mucosal growth. The molecular mechanisms regulating the effects of glutamine on intestinal cell growth and survival are poorly understood. Here, we show that addition of glutamine (1 mmol/l) enhanced rat intestinal epithelial (RIE)-1 cell growth; conversely, glutamine deprivation increased apoptosis as noted by increased DNA fragmentation and caspase-3 activity. To delineate signaling pathways involved in the effects of glutamine on intestinal cells, we assessed activation of extracellular signal-related kinase (ERK), protein kinase D (PKD), and phosphatidylinositol 3-kinase (PI3K)/Akt, which are important pathways in cell growth and survival. Addition of glutamine activated ERK and PKD in RIE-1 cells after a period of glutamine starvation; inhibition of ERK, but not PKD, increased cell apoptosis. Conversely, glutamine starvation alone increased phosphorylated Akt; inhibition of Akt enhanced RIE-1 cell DNA fragmentation. The role of ERK was further delineated using RIE-1 cells stably transfected with an inducible Ras. Apoptosis was significantly increased following ERK inhibition, despite Ras activation. Taken together, these results identify a critical role for the ERK signaling pathways in glutamine-mediated intestinal homeostasis. Furthermore, activation of PI3K/Akt during periods of glutamine deprivation likely occurs as a protective mechanism to limit apoptosis associated with cellular stress. Importantly, our findings provide novel mechanistic insights into the antiapoptotic effects of glutamine in the intestine.     

The cell survival signal Akt is differentially activated by PDGF-BB, EGF, and FGF-2 in osteoblastic cells

Stimulation of osteoblast survival signals may be an important mechanism of regulating bone anabolism. Protein kinase B (PKB/Akt), a serine-threonine protein kinase, is a critical regulator of normal cell growth, cell cycle progression, and cell survival. In this study we have investigated the signaling pathways activated by growth factors PDGF-BB, EGF, and FGF-2 and determined whether PDGF-BB, EGF, and FGF-2 activated Akt in human or mouse osteoblastic cells. The results demonstrated that both ERK1 and ERK2 were activated by FGF-2 and PDGF-BB. Activation of ERK1 and ERK2 by PDGF-BB and FGF-2 was inhibited by PD 098059 (100 microM), a specific inhibitor of MEK. Wortmannin (500 nM), a specific inhibitor of phosphatidylinositol 3-kinase ( PI 3-K), inhibited the activation of ERK1 and ERK2 by PDGF-BB but not by FGF-2 suggesting that PI 3-K mediated the activation of ERK MAPK pathway by PDGF-BB but not by FGF-2. Rapamycin, an inhibitor of p70 S6 protein kinase and a downstream target of ERK1/2 and PI 3-K, did not affect the activation of ERK1 and ERK2 by the growth factors. Furthermore, our results demonstrated that Akt, a downstream target of PI 3-K, was activated by PDGF-BB but not by FGF-2. Akt activation by PDGF-BB was inhibited by PI 3-kinase inhibitor LY294002. Rapamycin had no effect on Akt activation. Epidermal growth factor (EGF) also activated Akt in osteoblastic cells which was inhibited by LY294002 but not by rapamycin. Taken together, our data for the first time revealed that the activation of ERK1/2 by PDGF-BB is mediated by PI 3-K, and secondly, Akt is activated by PDGF-BB and EGF but not by FGF-2 in human and mouse osteoblastic cells. These results are of critical importance in understanding the role of these growth factors in apoptosis and cell survival. PDGF-BB and EGF but not FGF-2 may stimulate osteoblast cell survival.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.     

Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK.

Differentiated osteoclasts have a short life span. We tested various cytokines and growth factors for the effects on the survival of purified mature osteoclasts. In the absence of any added factors, osteoclasts exhibited the survival rate of less than 25% after a 24-h incubation. Among the tested factors, tumor necrosis factor-alpha (TNF-alpha) was found to increase the survival rate to approximately 80%. The TNF-alpha-enhanced survival of osteoclasts appeared to be associated with reduction in apoptosis and suppression of caspase activation. The antiapoptotic signaling pathways involved in the TNF-alpha-induced osteoclast survival were investigated. TNF-alpha treatment increased the phosphorylation of Akt in osteoclasts, which was suppressed by a phosphatidylinositol 3-kinase inhibitor LY294002 and an Src family kinase-selective inhibitor PP1. These inhibitors also attenuated the TNF-alpha stimulation of osteoclast survival. In addition an increase in the phosphorylation of ERK was observed upon TNF-alpha stimulation. PD98059, a specific inhibitor of the ERK-activating kinase MEK-1, abolished the TNF-alpha-induced ERK phosphorylation and osteoclast survival, and in these responses the involvement of Grb2 and ceramide was observed. These results suggest that TNF-alpha promotes the survival of osteoclasts by engaging the phosphatidylinositol 3-kinase Akt and MEK/ERK signaling pathways.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.     

IL-1alpha stimulation of osteoclast survival through the PI 3-kinase/Akt and ERK pathways.

Osteoclasts, cells that resorb bone, die once fully differentiated. Several factors including interleukin-1 (IL-1) have been shown to regulate the survival of mature osteoclasts. However, information on the mechanism underlying the regulation of osteoclast survival has been limited. In this study, we investigated the mechanism for the IL-1-stimulated survival of osteoclasts. Treatment of purified osteoclasts with IL-1alpha led to activation of the serine-threonine kinases Akt and ERK. Blocking the activation of Akt with LY294002, a specific inhibitor of the Akt up-stream molecule PI 3-kinase, or an with adenoviral vector for a dominant-negative form of Akt prevented the stimulation of osteoclast survival by IL-1alpha. PD98059, a specific inhibitor of the ERK-activating kinase MEK1, also abolished the effects of IL-1alpha on ERK activation and osteoclast survival. IL-1alpha reduced the apoptosis of osteoclasts by reducing caspase 3 activity. The IL-1alpha-mediated suppression of apoptosis was abolished by the PI 3-kinase/Akt or MEK1/ERK pathway inhibitor. These findings implicate the PI 3-kinase/Akt and ERK signaling pathways in the promotion of osteoclast survival by IL-1alpha.     

G beta gamma counteracts G alpha(q) signaling upon alpha(1)-adrenergic receptor stimulation

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.     

Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.     

The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons

Ras promotes robust survival of many cell systems by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, but little is understood about the survival functions of the Ras/ERK pathway. We have used three different effector-loop mutant forms of Ras, each of which activates a single downstream effector pathway, to dissect their individual contributions to survival of nerve growth factor (NGF)-dependent sympathetic neurons. The PI3-kinase pathway-selective protein Ras(Val-12)Y40C was as powerful as oncogenic Ras(Val-12) in preventing apoptosis induced by NGF deprivation but conferred no protection against apoptosis induced by cytosine arabinoside. Identical results were obtained with transfected Akt. In contrast, the ERK pathway-selective protein Ras(Val-12)T35S had no protective effects on NGF-deprived neurons but was almost as strongly protective as Ras(Val-12) against cytosine arabinoside-induced apoptosis. The protective effects of Ras(Val-12)T35S against cytosine arabinoside were completely abolished by the ERK pathway inhibitor PD98059. Ras(Val-12)E37G, an activator of RalGDS, had no survival effect on either death pathway, similar to RasS17N, the full survival antagonist. Thus, Ras provides two independent survival pathways each of which inhibits a distinct apoptotic mechanism. Our study presents one of the few clear-cut cases where only the Ras/ERK, but not the Ras/PI3K/Akt pathway, plays a dominant survival signaling role.     

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.     

Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways

The function of muscarinic acetylcholine receptors expressed in oligodendrocytes and in myelin has remained largely undetermined. Here we present evidence that incubation of oligodendrocyte progenitors, deprived of growth factor, with the acetylcholine analog carbachol significantly reduced cell death by apoptosis and blocked caspase-3 cleavage. This protective effect was reversed by atropine, a muscarinic acetylcholine receptor antagonist, as well as by specific inhibitors of intracellular signaling molecules, including phosphatidylinositol 3-kinase (Wortmannin and LY294002), Akt (Akt inhibitor III) and Src-like tyrosine kinases (PP2), but not by the mitogen-activated protein kinase kinase inhibitor, PD98059. Activation of Akt by carbachol was antagonized by atropine and inhibited by LY294002 and PP2. The Src-like tyrosine kinase inhibitor, PP2, also reduced carbachol stimulation of extracellular signal-regulated kinases 1/2 and cAMP-response element binding protein in a dose-dependent manner. Furthermore, carbachol increased tyrosine-phosphorylation of Fyn, a member of the Src-like tyrosine kinases. These results indicate that muscarinic acetylcholine receptors play an important role in oligodendrocyte progenitor survival through transduction pathways involving activation of Src-like tyrosine kinases and phosphatidylinositol 3-kinase/Akt.     

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.     

TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src

In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt.     

Glial cell line-derived neurotrophic factor-stimulated phosphatidylinositol 3-kinase and Akt activities exert opposing effects on the ERK pathway: importance for the rescue of neuroectodermic cells

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in rescuing neural crest cells from apoptosis during their migration in the foregut. This survival factor binds to the heterodimer GDNF family receptor alpha1/Ret, inducing the Ret tyrosine kinase activity. ret loss-of-function mutations result in Hirschsprung's disease, a frequent developmental defect of the enteric nervous system. Although critical to enteric nervous system development, the intracellular signaling cascades activated by GDNF and their importance in neuroectodermic cell survival still remain elusive. Using the neuroectodermic SK-N-MC cell line, we found that the Ret tyrosine kinase activity is essential for GDNF to induce phosphatidylinositol 3-kinase (PI3K)/Akt and ERK pathways as well as cell rescue. We demonstrate that activation of PI3K is mandatory for GDNF-induced cell survival. In addition, evidence is provided for a critical up-regulation of the ERK pathway by PI3K at the level of Raf-1. Conversely, Akt inhibits the ERK pathway. Thus, both PI3K and Akt act in concert to finely regulate the level of ERK. We found that Akt activation is indispensable for counteracting the apoptotic signal on mitochondria, whereas ERK is partially involved in precluding procaspase-3 cleavage. Altogether, these findings underscore the importance of the Ret/PI3K/Akt pathway in GDNF-induced neuroectodermic cell survival.     

Inhibition of TNF-alpha-induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate is mediated by the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways up-stream of caspase 3

The role of protein kinases in the inhibition of TNF-alpha associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-alpha and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-alpha produced only a modest and delayed activation of Akt. In the presence of TNF-alpha, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-alpha itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 microM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-alpha. Furthermore, the inhibition of the Mek1/Mek2-->ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-alpha-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.