Role of extracellular signal-regulated kinase and phosphatidylinositol-3 kinase in chemoattractant and LPS delay of constitutive neutrophil apoptosis

The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.     

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.     

Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways

Cartducin, a paralog of Acrp30/adiponectin, is a secretory protein produced by both chondrogenic precursors and proliferating chondrocytes, and belongs to a novel C1q family of proteins. We have recently shown that cartducin promotes the growth of both mesenchymal chondroprogenitor cells and chondrosarcoma-derived chondrocytic cells in vitro. However, the cartducin-signaling pathways responsible for the regulation of cell proliferation have not been documented. In this study, we examined whether cartducin exists in serum and further investigated the intracellular signaling pathways stimulated by cartducin in mesenchymal chondroprogenitor cells. Western blot analysis showed that, unlike Acrp30/adiponectin, cartducin was undetectable in mouse serum. Next, mesenchymal chondroprogenitor N1511 cells were stimulated with cartducin, and three major groups of mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway were examined. Cartducin activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, but not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1/2 inhibitor, U0126, blocked cartducin-stimulated ERK1/2 phosphorylation and suppressed the DNA synthesis induced by cartducin in N1511 cells. The PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphorylation and a decrease in cartducin-induced DNA synthesis in N1511 cells was also observed. These data suggest that cartducin is a peripheral skeletal growth factor, and that the proliferation of mesenchymal chondroprogenitor cells stimulated by cartducin is associated with activations of the ERK1/2 and PI3K/Akt signaling pathways.     

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.     

Cyclic AMP inhibits extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways by inhibiting Rap1

Cyclic AMP inhibited both ERK and Akt activities in rat C6 glioma cells. A constitutively active form of phosphatidylinositol 3-kinase (PI3K) prevented cAMP from inhibiting Akt, suggesting that the inactivation of Akt by cAMP is a consequence of PI3K inhibition. Neither protein kinase A nor Epac (Exchange protein directly activated by cAMP), two known direct effectors of cAMP, mediated the cAMP-induced inhibition of ERK and Akt phosphorylation. Cyclic AMP inhibited Rap1 activation in C6 cells. Moreover, inhibition of Rap1 by a Rap1 GTPase-activating protein-1 also resulted in a decrease in ERK and Akt phosphorylation, which was not further decreased by cAMP, suggesting that cAMP inhibits ERK and Akt by inhibiting Rap1. The role of Rap1 in ERK and Akt activity was further demonstrated by our observation that an active form of Epac, which activated Rap1 in the absence of cAMP, increased ERK and Akt phosphorylation. Inhibition of ERK and/or PI3K pathways mediated the inhibitory effects of cAMP on insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 gene expression. Moreover, cAMP, as well as ERK and PI3K inhibitors produced equivalent stimulation and inhibition, respectively, of p27(Kip1) and cyclin D2 protein levels, potentially explaining the observation that cAMP prevented C6 cells from entering S phase.     

Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

Apoptosis is a form of programmed cell death that plays a pivotal role during development and in the homeostasis of the adult nervous systems. However, mechanisms that regulate neuronal apoptosis are not well defined. Here, we report that brain-derived neurotrophic factor (BDNF) protects cortical neurons against apoptosis induced by camptothecin or serum deprivation and activates the extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Using pharmacological agents and transient transfection with dominant interfering or constitutive active components of the ERK or the PI 3-kinase pathway, we demonstrate that the ERK pathway plays a major role in BDNF neuroprotection against camptothecin. Furthermore, ERK is activated in cortical neurons during camptothecin-induced apoptosis, and inhibition of ERK increases apoptosis. In contrast, the PI 3-kinase pathway is the dominant survival mechanism for serum-dependent survival under normal culture conditions and for BDNF protection against serum withdrawal. These results suggest that the ERK pathway is one of several neuroprotective mechanisms that are activated by stress to counteract death signals in central nervous system neurons. Furthermore, the relative contribution of the ERK and PI 3-kinase pathways to neuronal survival may depend on the type of cellular injury.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

Luteolin inhibits pyrogallol-induced apoptosis through the extracellular signal-regulated kinase signaling pathway

Luteolin is an antioxidative, antitumor and anti-inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol-induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0-400 μm) dose-dependently induced reactive oxygen species production, cytotoxicity, an annexin V-fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78-50 μm) in a concentration-dependent manner. Luteolin suppressed the poly (ADP-ribose) polymerase activation, caspase-8 cleavage and p38 mitogen-activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal-regulated kinase signaling pathway to counteract the pyrogallol-induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress-induced apoptosis via the extracellular signal-regulated kinase signaling pathway.     

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.     

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase,extracellular signal-regulated kinase and protein kinase C signaling pathways

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.     

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.     

Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca(2+) channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca(2+) channel blocker, and chelation of extracellular Ca(2+). These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.     

Differential signalling of purinoceptors in HeLa cells through the extracellular signal-regulated kinase and protein kinase C pathways

We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities.     

Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways.

Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.     

Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

Co-induction of cell death and survival pathways by phosphoinositide 3-kinase

A single stimulus can induce both the cell death and survival pathway, suggesting that these pathways share common upstream signaling components. In order to define these components, human U937 cells grown in 10% serum were exposed to serum-free media. This treatment resulted in apoptosis, which was found to be mediated by SAPK/JNK. It was previously reported that the serum withdrawal (SW)-induced SAPK activation is mediated by a positive mutual interaction between the reactive oxygen species (ROS) and phosphoinositide 3-kinase (PI3K). This study shows that the ROS/PI3K interaction also induces a NF-kappaB-dependent survival pathway. Despite the role of PI3K, Akt was found to be irrelevant to the activation of SAPK and NF-kappaB. Comparative analyses of SAPK and NF-kappaB for their responses to exogenous H(2)O(2) revealed that SAPK activation requires much higher H(2)O(2) concentrations than those required for NF-kappaB activation. Moreover, high lethal concentrations of H(2)O(2) were found to activate NF-kappaB and SAPK in a PI3K-independent manner. These results suggest that ROS induce both the SAPK-dependent apoptotic and NF-kappaB-mediated survival pathways, and these inducer signals are amplified by PI3K in the SW-triggered pathway. Cell death appears to be favored as this amplification proceeds.     

Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC- signaling pathways.     

Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor

In vitro addition of stem cell factor (SCF) to c-kit-expressing A(1)-A(4) spermatogonia from prepuberal mice stimulates their progression into the mitotic cell cycle and significantly reduces apoptosis in these cells. SCF addition results in a transient activation of extracellular signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase. These events are followed by a rapid re-distribution of cyclin D3, which becomes predominantly nuclear, whereas its total cellular amount does not change. Nuclear accumulation of cyclin D3 is coupled to transient activation of the associated kinase activity, assayed using the retinoblastoma protein (Rb) as a substrate. These events were followed by a transient accumulation of cyclin E, stimulation of the associated histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb hyper-phosphorylation. All the events associated with SCF-induced cell cycle progression are inhibited by the addition of either a PI3K inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor, indicating that both MEK and PI3K are essential for c-kit-mediated proliferative response. On the contrary, the anti-apoptotic effect of SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways.