Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.     

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.     

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.     

All-trans-retinoid acid induces the differentiation of P19 cells into neurons involved in the PI3K/Akt/GSK3β signaling pathway

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.     

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma × glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the “pro-survival” kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma × glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen^2,5]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G_i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G_q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.     

Insulin blocks cytochrome c release in the reperfused brain through PI3-K signaling and by promoting Bax/Bcl-XL binding

The critical event of the intrinsic pathway of apoptosis following transient global brain ischemia is the release of cytochrome c from the mitochondria. In vitro studies have shown that insulin can signal specifically via phosphatidylinositol-3-OH-kinase (PI3-K) and Akt to prevent cytochrome c release. Therefore, insulin may exert its neuroprotective effects during brain reperfusion by blocking cytochrome c release. We hypothesized that insulin acts through PI3-K, Akt, and Bcl-2 family proteins to inhibit cytochrome c release following transient global brain ischemia. We found that a single bolus of insulin given immediately upon reperfusion inhibited cytochrome c release for at least 24 h, and produced a fivefold improvement in neuronal survival at 14 days. Moreover, insulin's ability to inhibit cytochrome c release was completely dependent on PI3-K signaling and insulin induces phosphorylation of Akt through PI3-K. In untreated animals, there was an increase in mitochondrial Bax at 6 h of reperfusion, and Bax binding to Bcl-X_L was disrupted at the mitochondria. Insulin prevented both these events in a PI3-K-dependent manner. In summary, insulin regulates cytochrome c release through PI3-K likely by activating Akt, promoting the binding between Bax and Bcl-X_L, and by preventing Bax translocation to the mitochondria.     

Hydrogen sulfide improves left ventricular function in smoking rats via regulation of apoptosis and autophagy

The present study was designed to investigate the protective effects of hydrogen sulfide (H₂S) against cigarette smoking-induced left ventricular dysfunction in rats. Left ventricular structure and function were assessed using two-dimensional echocardiography. Cardiomyocyte apoptosis was determined by Annexin V/PI and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our results indicated that administration of NaHS (a donor of H₂S) could protect against smoking-induced left ventricular systolic dysfunction. H₂S was found to exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 mitogen-activated protein kinases pathways and activating PI3K/Akt signaling. Moreover, H₂S could also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H₂S can improve left ventricular systolic function in smoking rats via regulation of apoptosis and autophagy.     

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.     

Phosphatidylinositol 3-kinase signaling to Akt mediates survival in isolated canine islets of Langerhans

The isolation of islet cells from the pancreas by enzymatic digestion causes many of these cells to undergo apoptosis. The aim of this work was to investigate the role of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling in mediating the survival of isolated islets. Insulin-like growth factor-1 (IGF-I) was examined as a potential culture media supplement that could rescue isolated islets from their apoptotic fate. Western blot analysis demonstrated that Akt phosphorylation peaks 20 h after routine islet isolation. PI3-K inhibition with wortmannin abolished both basal and IGF-I-mediated Akt phosphorylation. IGF-I did not increase survival of isolated islets under normal conditions but it did have a protective effect against cytokine (TNF-alpha, IL-1beta, INF-gamma)-mediated cell death. The protective effect of IGF-I against cytokine-stimulated apoptosis was blocked by wortmannin. In addition, inhibition of basal levels of PI3-K activity caused a 31% decrease in islet survival, as shown by MTT assay. These results demonstrate that the PI3-K/Akt pathway mediates survival of isolated islets of Langerhans.     

Effect of exogenous surfactant on phosphatidylinositol 3-kinase-Akt pathway and peroxisome proliferator activated receptor-γ during endotoxin induced acute respiratory distress syndrome

Lipopolysaccharide induced acute respiratory distress syndrome (ARDS) leads to an unacceptably high mortality. In this regard, the anti-inflammatory properties of surfactant may provide a therapeutic option. Phosphoinositide 3-kinase (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. This study explores the mechanisms of surfactant dependent protection by regulating PPAR-γ in a rat model of ARDS. Sprague-Dawley male rats were divided into four groups: buffer controls; rats challenged with LPS (055:B5 E. coli); challenged with LPS and treated with porcine surfactant; and challenged with LPS and treated with synthetic surfactant. Expression of PI3-K, Akt, GSK3-β, and PPAR-γ were studied by western immunoblot, immunofluorescence and by immunohistochemistry. In vivo endotoxin administration to rat resulted in activation of PI3-K and Akt in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in rat with surfactant administration. Similar results were also seen in PPAR-γ expression. These results show that PI3-K occupies a central position in regulating endotoxin-induced ALI involving inflammatory responses. Surfactant treatment conferred protection in rat model dependent on PPAR-γ and inhibition of PI3-K/Akt pathway.     

ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.     

Phosphatidylinositol 3-Kinase γ Is Required for the Development of Experimental Cerebral Malaria

Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ^-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ^-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ^-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3⁺CD8⁺ T cells were significantly reduced in the brain of infected PI3Kγ^-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ^-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.