Epstein-Barr virus latent membrane protein 2A mediates transformation through constitutive activation of the Ras/PI3-K/Akt Pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells

Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV) oncoprotein, mimics a constitutively activated tumor necrosis factor receptor and activates various signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt. LMP1 is essential for EBV-mediated B-cell transformation and is sufficient to transform several cell lines. Cellular transformation has been associated strongly with genomic instability, while DNA repair plays an important role in maintaining genomic stability. Previously, we have shown that LMP1 represses DNA repair by the C-terminal activating region 1 (CTAR1) in human epithelial cells. In the present study, we demonstrate that the PI3K/Akt pathway is required for LMP1-mediated repression of DNA repair. Through the LMP1/PI3K/Akt pathway, FOXO3a, which can induce DNA repair, is inactivated because of phosphorylation and relocalization. Expression of a constitutively active FOXO3a mutant can rescue LMP1-mediated repression of DNA repair. Furthermore, LMP1 can decrease the expression of DNA damage-binding protein 1 (DDB1), which functions in nucleotide excision repair, through the PI3K/Akt/FOXO3a pathway. LMP1-mediated repression of DNA repair is restored by DDB1, although only partially. These results suggest that LMP1 triggers the PI3K/Akt pathway to inactivate FOXO3a and decrease DDB1, which can lead to repression of DNA repair and may contribute to genomic instability in human epithelial cells.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Latent membrane protein 2A-mediated effects on the phosphatidylinositol 3-Kinase/Akt pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membranes of B lymphocytes and blocks B-cell receptor (BCR) signaling in EBV-transformed B lymphocytes in vitro. The phosphotyrosine motifs at positions 74 or 85 and 112 within the LMP2A amino-terminal domain are essential for the LMP2A-mediated block of B-cell signal transduction. In vivo studies indicate that LMP2A allows B-cell survival in the absence of normal BCR signals. A possible role for Akt in the LMP2A-mediated B-cell survival was investigated. The protein kinase Akt is a crucial regulator of cell survival and is activated within B lymphocytes upon BCR cross-linking. LMP2A expression resulted in the constitutive phosphorylation of Akt, and this LMP2A effect is dependent on phosphatidylinositol 3-kinase activity. In addition, recruitment of Syk and Lyn protein tyrosine kinases (PTKs) to tyrosines 74 or 85 and 112, respectively, are critical for LMP2A-mediated Akt phosphorylation. However, the ability of LMP2A to mediate a survival phenotype downstream of Akt could not be detected in EBV-negative Akata cells. This would indicate that LMP2A is not responsible for EBV-dependent Burkitt's lymphoma cell survival.     

MIP-1alpha induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration.

The chemokine macrophage inflammatory protein-1 alpha [MIP-1alpha] causes migration of B cells and also induces changes in antibody secretion. However, the signal transduction pathways leading to these phenotypic changes remain undefined. We have identified a signal transduction pathway initiated by MIP-1alpha in B cells. Here we report that stimulation of tonsil B cells with MIP-1alpha induces phosphatidylinositol 3-kinase [PI3-K] activation. Kinase activity was transient with peak induction occurring within 2.5 to 5 min after stimulation and was dose-dependent. In addition stimulation with MIP-1alpha induces tyrosine phosphorylation of the proline-rich tyrosine kinase Pyk-2. Immunoprecipitation analysis showed a constitutive association between Pyk-2 and PI3-K and pretreatment of MIP-1alpha-stimulated B cells with wortmannin, a specific inhibitor of PI3-K, resulted in a loss of PI3-K activity. The PI3-K inhibitor wortmannin prevented B cells from migrating in response to MIP-1alpha. Hence, PI3-K and Pyk-2 seem to be components of a signal transduction pathway induced by stimulation of B cells with MIP-1alpha, and this pathway may play a role in B-cell migration.     

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.     

Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells

Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.     

Grb2 associated binder 2 couples B-cell receptor to cell survival

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.     

Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation

The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction.     

Unique signaling properties of CTAR1 in LMP1-mediated transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.     

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1

Apoptosis is an important cell suicide program which involves the caspases activation and is implicated in physiological and pathological processes. Poly(ADP-ribose) polymerase (PARP) cleavage is often associated with apoptosis and has been served as one hallmark of apoptosis and caspase activation. In this study, we aimed to determine TGF-beta1-induced apoptosis and to examine the involvement of caspases and its relationship with PARP cleavage. TGF-beta1 induces strong apoptosis of AML-12 cells which can be detected by DNA fragmentation, FACS, and morphological assays. Z-VAD-fmk, a selective caspase inhibitor, partially inhibits the TGF-beta1-induced apoptosis; but has no effect on TGF-beta1-induced DNA fragmentation and PARP cleavage. However, BD-fmk, a broad-spectrum caspase inhibitor, completely suppresses TGF-beta1-induced apoptosis, but unexpectedly does not inhibit TGF-beta1-induced PARP cleavage. Furthermore, Z-VAD-fmk treatment is able to completely inhibit the daunorubicin-induced apoptosis in A-431 cells, but only slightly blocks the daunorubicin-induced PARP cleavage, whereas BD-fmk can inhibit both daunorubicin-induced apoptosis and PARP cleavage completely. In addition, we observed that both TGF-beta1-induced apoptosis and PARP degradation in AML-12 cells can be completely blocked by inhibiting the protein synthesis with cycloheximide. These results demonstrate for the first time that TGF-beta1-induced caspase-dependent apoptosis is associated with caspase-independent PARP cleavage that requires the TGF-beta1-induced synthesis of new proteins. The results indicate that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation. The results also suggest that PARP cleavage may occur as an independent event that can be disassociated with cell apoptosis.     

Role of PI3-kinase in isoproterenol and IGF-1 induced ecNOS activity

Phosphatidylinositol 3-kinase (PI3-K) has been shown to mediate insulin and insulin-like growth factor-1 (IGF-1)-induced nitric oxide (NO) generation and, thus, vascular tone. A role for PI3-K in G-protein-coupled receptor signal transduction has been reported. As beta (beta2)-adrenergic vascular actions are partly dependent on NO, we have investigated the role of PI3-K in isoproterenol (Iso) and IGF-1 induced endothelial NO synthase (ecNOS) activity in rat aortic endothelial cells (RAEC). Cell lysates of RAEC, exposed to Iso (10 micromol/L) for 5 min and 6 h, and to IGF-1 (100 nM) for 10 min and 6 h, or pretreated with PI3-K inhibitor Wortmannin (WT), were used for measuring PI3-K activity, p85kDa regulatory protein, and citrulline production. Results show that Iso and IGF-1 increased a p85 subunit and citrulline production, and also enhanced 32P incorporation into PIP3. Pretreatment with WT inhibited Iso-stimulated ecNOS, as well as, PI3-K activity. Iso enhanced association of ecNOS with the triton X-100-insoluble fraction of RAEC. These data indicate that the endothelial cell PI3-K pathway mediates, in part, the release of NO and subsequent vasorelaxation in response to this beta-agonist, as well as, IGF-1.     

A truncated kringle domain of human apolipoprotein(a) inhibits the activation of extracellular signal-regulated kinase 1 and 2 through a tyrosine phosphatase-dependent pathway

Most proangiogenic factors exert their biological effects primarily by activating extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt signaling pathways. These pathways appear to play a critical role in endothelial cell migration, because selective inhibition of either ERK or PI3-K/Akt signaling almost completely prevented endothelial cell migration. Recently, we demonstrated that a truncated kringle domain of human apolipoprotein(a), termed rhLK68, inhibits endothelial cell migration in vitro. However, its mechanism of action was not well defined. In this study, we determined the effects of rhLK68 on ERK1/2 and PI3-K/Akt signaling pathways to explore the molecular mechanism of rhLK68-mediated inhibition of endothelial cell migration. Treatment with rhLK68 inhibited ERK1/2 phosphorylation but did not influence Akt activation. Interestingly, an inhibitor of protein-tyrosine phosphatase, sodium orthovanadate, dose-dependently reversed both rhLK68-induced dephosphorylation of ERK1/2 and decreased migration of endothelial cells, whereas rhLK68 showed no significant effects on MEKs phosphorylation. In conclusion, these results indicate that inhibition of endothelial cell migration by rhLK68 may be achieved by interfering with ERK1/2 activation via a protein-tyrosine phosphatase-dependent pathway.     

Transforming growth factor-beta activates both pro-apoptotic and survival signals in fetal rat hepatocytes

Transforming growth factor-beta (TGF-beta) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives concomitant with changes in morphology and phenotype, reminiscent of an epithelial mesenchymal transition (EMT) [Exp. Cell Res. 252 (1999) 281-291]. In this work, we have isolated the subpopulation that survives to TGF-beta-induced apoptosis, showing that these cells maintain the response to TGF-beta in terms of Smads activation and growth inhibition. Analyses of the intracellular signals that could impair the apoptotic effects of TGF-beta have indicated that the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway is activated in these resistant cells. Experiments in fetal rat hepatocytes have shown that TGF-beta is able to transiently activate PI 3-K/Akt by a mechanism independent of protein synthesis but dependent on a tyrosine kinase activity. Pro-apoptotic signals, such as oxidative stress and caspases, contribute to the loss of Akt at later times. Inhibiting PI 3-K sensitizes fetal hepatocytes (FH) to the apoptosis induced by TGF-beta and causes spontaneous death in the resistant cells. In conclusion, TGF-beta, as it is known for other cytokines, could be inducing pro-apoptotic and survival signals in hepatocytes, the balance among them will decide cell fate.