Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.     

7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.

BACKGROUND: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis. METHODS: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. RESULTS: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures. CONCLUSIONS: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.     

Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.     

Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP-4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC

BMP-4 and BMP-7 are associated with the suppression of granulosa cell apoptosis. LY294002 (PI3K inhibitor) or UCN-01 (PDK-1 inhibitor) increased the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. The inhibitors of ERK and p38 (SB203580) did not increase the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. Akt inhibitor did not induce apoptosis in the BMP-4-treated granulosa cells, whereas it did induce apoptosis of the BMP-7-treated granulosa cells. In the granulosa cells treated with BMP-4, the PKC inhibitor increased the percentage of apoptotic cells. Our data show that BMP-4 and BMP-7 are associated with granulosa cell survival via several non-Smad specific pathways: BMP-4 via the PI3K/PDK-1/PKC and BMP-7 via the PI3K/PDK-1/Akt.     

Regulation of Ang2 release by PTEN/PI3-kinase/Akt in lung microvascular endothelial cells

Angiopoietin-2 (Ang2) is a Tie-2 ligand that destabilizes vascular structures, allowing for neovascularization or vessel regression depending on local vascular endothelial cell growth factor (VEGF) concentrations. Although various stimuli have been shown to affect Ang2 expression, information on the underlying mechanisms involved in Ang2 production in endothelial cells (EC) is just beginning to emerge. In the present study, we have used adenovirus-mediated gene transfer and pharmacological inhibitors to examine the role of the PTEN/PI3-K/Akt pathway on Ang2 release. Inhibition of PI3-kinase with wortmannin led to a stimulation of basal Ang2 release in EC, while overexpression of an active form of Akt reduced Ang2. In addition, adenovirus-mediated gene transfer of the phosphatase PTEN stimulated Ang2 release. Incubation of the cells with Ang1, an agent that activates the PI3-K/Akt pathway in EC, reduced Ang2 release. This effect of Ang1 could be prevented by wortmannin and LY-294002 pretreatment. Similarly, in VEGF-treated EC the increase in Ang2 production observed was greater in the presence of a PI3-K inhibitor. Our observations that PTEN acts as a positive modulator of Ang2 release, while activation of the PI3-K/Akt pathway downregulates Ang2, reveal an additional mechanism through which the PTEN/PI3-K/Akt pathway could affect the angiogenic process.     

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.     

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.     

TNF-alpha induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-alpha and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-alpha resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-alpha induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-alpha was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-alpha activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-alpha-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-alpha in cardiac myocytes.     

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G_βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.     

Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages

7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of cPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388D1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.     

Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis

We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.     

The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells

Activation of Lyn, a Src-related nonreceptor tyrosine kinase, in trophoblast cells is associated with trophoblast giant cell differentiation. The purpose of the present work was to use Lyn as a tool to identify signaling pathways regulating the endocrine differentiation of trophoblast cells. The Src homology 3 domain of Lyn was shown to display differentiation-dependent associations with other regulatory proteins, including phosphatidylinositol 3-kinase (PI3-K). PI3-K activation was dependent upon trophoblast giant cell differentiation. The downstream mediator of PI3-K, Akt/protein kinase B, also exhibited differentiation-dependent activation. Lyn is a potential regulator of the PI3-K/Akt signaling pathway, as are receptor tyrosine kinases. Protein tyrosine kinase profiling was used to identify two candidate regulators of the PI3-K/Akt pathway, fibroblast growth factor receptor-1 and Sky. At least part of the activation of Akt in differentiating trophoblast giant cells involves an autocrine growth arrest-specific-6-Sky signaling pathway. Inhibition of PI3-K activities via treatment with LY294002 disrupted Akt activation and interfered with the endocrine differentiation of trophoblast giant cells. In summary, activation of the PI3-K/Akt signaling pathway regulates the development of the differentiated trophoblast giant cell phenotype.     

Protective effects of docosahexaenoic acid in staurosporine-induced apoptosis: involvement of phosphatidylinositol-3 kinase pathway

Docosahexaenoic acid (22:6n-3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kim et al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase-3 activity, cleavage of pro-caspase-3 to active caspase-3, DNA strand-breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3-kinase (PI3-K) inhibitors, wortmanin, and LY-294002 abolished the protective effect of DHA in ST-induced apoptosis. Concurrently, ST-treatment significantly decreased the phosphorylation status of Akt at Ser-473 and Thr-308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time-dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine-free medium, the PS increase diminished and the DHA effect on caspase-3 activation as well as Akt phosphorylation in ST-induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST-induced apoptosis in a PS- and PI3-K-dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3-K/Akt pathway, facilitated by DHA-induced PS accumulation.     

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.     

PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration

This study was designed to identify the molecular mechanisms of phosphatidylinositol 3-kinase (PI3K)-induced actin filament remodeling and cell migration. Expression of active forms of PI3K, v-P3k or Myr-P3k, was sufficient to induce actin filament remodeling to lead to an increase in cell migration, as well as the activation of Akt in chicken embryo fibroblast (CEF) cells. Either the inhibition of PI3K activity using a PI3K-specific inhibitor, LY-294002, or the disruption of Akt activity restored the integrity of actin filaments in CEF cells and inhibited PI3K-induced cell migration. We also found that expression of an activated form of Akt (Myr-Akt) was sufficient to remodel actin filaments to lead to an increase in cell migration, which was unable to be inhibited by the presence of LY-294002. Furthermore, we found that p70S6K1 kinase was a downstream molecule that can mediate the effects of both PI3K and Akt on actin filaments and cell migration. Overexpression of an active form of p70S6K1 was sufficient to induce actin filament remodeling and cell migration in CEF cells, which requires Rac activity. These results demonstrate that activation of PI3K activity alone is sufficient to remodel actin filaments to increase cell migration through the activation of Akt and p70S6K1 in CEF cells. phosphatidylinositol 3-kinase; Rac; actin filaments     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.