Contribution of the PI 3-kinase/Akt survival pathway toward osmotic preconditioning

Osmolytes are rapidly lost from the ischemic heart, an effect thought to benefit the heart by reducing the osmotic load. However, the observation that chronic lowering of one of the prominent osmolytes, taurine, is more beneficial to the ischemic heart than acute taurine loss suggests that osmotic stress may benefit the ischemic heart through multiple mechanisms. The present study examines the possibility that chronic osmotic stress preconditions the heart in part by stimulating a cardioprotective, osmotic-linked signaling pathway. Hyperosmotic stress was produced by treating rat neonatal cardiomyocytes during the pre-hypoxic period with either the taurine depleting agent, beta-alanine (5 mM), or with medium containing 25 mM mannitol. The cells were then subjected to chemical hypoxia in medium containing 3 mM Amytal and 10 mM deoxyglucose but lacking beta-alanine and mannitol. Cells that had been pretreated with either 5 mM beta-alanine or 25 mM mannitol exhibited resistance against hypoxia-induced apoptosis and necrosis. Associated with the osmotically preconditioned state was the activation of Akt and the inactivation of the pro-apoptotic factor, Bad, both events blocked by the inhibition of PI 3-kinase. However, preconditioning the cardiomyocyte with mannitol had no effect on the generation of free radicals during the hypoxic period. Osmotic stress also promoted the upregulation of the anti-apoptotic factor, Bcl-2. Since inhibition of PI 3-kinase with Wortmannin also prevents osmotic-mediated cardioprotection, we conclude that hyperosmotic-mediated activation of the PI 3-kinase/Akt pathway contributes to osmotic preconditioning.     

Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.     

Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ~50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Regulatory Roles of the PI3K/Akt Signaling Pathway in Rats with Severe Acute Pancreatitis

The phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) pathway plays a key role in inflammation. However, the regulatory roles of PI3K/Akt in severe acute pancreatitis (SAP) have not been elucidated. The aim of this study was to investigate the impact of wortmannin, a PI3K/Akt inhibitor, on SAP rats through exposure to sodium taurocholate (STC) after 3 h and 6 h. The SAP group was found to have a significant increase in pancreas Akt expression, along with the activation of serum amylase, TNF-α, IL-1β, and IL-6, and pancreas histological aggravation. The administration of wortmannin in SAP rats reduced Akt expression, attenuated the level of serum amylase and inflammation factor, and alleviated the damage of pancreatic tissue. Furthermore, the administration of wortmannin led to an obvious reduction in NF-κB and p38MAPK expression in SAP rats. These findings showed that the PI3K/Akt inhibitor wortmannin decreases inflammatory cytokines in SAP rats and suggests its regulatory mechanisms may occur through the suppression on NF-κB and p38MAPK activity.     

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway.     

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.     

FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line

Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.     

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.     

Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl-D-aspartate receptor-mediated Akt1 activation

Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.     

p53 down-regulation: a new molecular mechanism involved in ischaemic preconditioning

Ischaemic preconditioning is associated with the activation of prosurvival mechanisms. Here we demonstrate that following a preconditioning protocol, the proapoptotic p53 is inactivated possibly via phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-murine double minute 2 (Mdm2) phosphorylation. Our data show that in preconditioned hearts Mdm2 was significantly phosphorylated, and wortmannin (a PI3K inhibitor) abrogated this effect (Western blotting). Also in preconditioned hearts p53 was shown to be bound to phospho-Mdm2 (co-immunoprecipitation). Furthermore, pifithrin alpha (a p53 inhibitor), administered to isolated perfused hearts prior to ischaemia, significantly attenuated the infarction. In conclusion our results suggest that p53 is implicated in ischaemia/reperfusion injury and that preconditioning counterbalances this effect via PI3K-Akt-Mdm2 phosphorylation.     

Targeting Class IA PI3K Isoforms Selectively Impairs Cell Growth,Survival, and Migration in Glioblastoma

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.     

Syk is required for the activation of Akt survival pathway in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H(2)O(2). In Syk-deficient DT40 cells treated with low doses of H(2)O(2) (10-100 microm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H(2)O(2), a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H(2)O(2), was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.     

Molecular Mechanism behind Rotavirus NSP1-Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85 Subunit of PI3 Kinase

Our previous study had reported on the interaction of rotavirus NSP1 with cellular phosphoinositide 3-kinase (PI3K) during activation of the PI3K pathway (P. Bagchi et al., J. Virol. 84:6834–6845, 2010). In this study, we have analyzed the molecular mechanism behind this interaction. Results showed that this interaction is direct and that both α and β isomers of the PI3K regulatory subunit p85 and full-length NSP1 are important for this interaction, which results in efficient activation of the PI3K/Akt pathway during rotavirus infection.     

Ischemic preconditioning protects by activating prosurvival kinases at reperfusion

Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.