Regulation of survivin by PI3K/Akt/p70S6K1 pathway

PI3K activation is commonly observed in many human cancer cells. Survivin expression is elevated in cancer cells, and induced by some growth factors through PI3K activation. However, it is not clear whether PI3K activation is sufficient to induce survivin expression. To investigate the role of PI3K pathway in the regulation of survivin, we expressed an active form of PI3K, v-P3k in chicken embryonic fibroblast cells (CEF), and found that overexpression of PI3K-induced survivin mRNA expression. Forced expression of wild-type but not mutant tumor suppressor PTEN in CEF decreased survivin mRNA levels. PI3K regulates survivin expression through Akt activation. To further investigate downstream target of PI3K and Akt in regulating the expression of survivin mRNA, we found that PI3K and Akt-induced p70S6K1 activation and that overexpression of p70S6K1 alone was sufficient to induce survivin expression. The treatment of CEF cells by rapamycin decreased the survivin mRNA expression. This result demonstrated that p70S6K1 is an important target downstream of PI3K and Akt in regulating suvivin mRNA expression. The knockdown of survivin mRNA expression by its specific siRNA induced apoptosis of cancer cells when the cells were treated with LY294002 or taxol. Taken together, these results demonstrated that PI3K/Akt/p70S6K1 pathway is essential for regulating survivin mRNA expression.     

Regulation of mTOR and p70 S6 kinase by the muscarinic M4 receptor in PC12 cells

The G_i-coupled M₄ muscarinic acetylcholine receptor (mAChR) has recently been shown to stimulate the survival of PC12 cells through the PI3K/Akt/tuberin pathway. Since mTOR and p70S6K are critical components in activating translation which lie downstream of tuberin, we examined the ability of M₄ mAChR to regulate these targets in PC12 cells. Carbachol (CCh) dose-dependently stimulated both mTOR and p70S6K phosphorylations and these responses were abolished by pertussis toxin pretreatment, indicating the involvement of the G_i-coupled M₄ mAChR. Phosphorylations of both mTOR and p70S6K were effectively blocked upon inhibition of PI3K by wortmannin. As compared to similar responses elicited by the nerve growth factor (NGF), the M₄ mAChR-induced activation of Akt/tuberin/mTOR/p70S6K occurred in a relatively transient manner. Although inhibition of protein phosphatase 2A by okadaic acid augmented the transient effects of CCh on Akt/tuberin phosphorylations, it failed to significantly prolong these responses. The total protein level of PTEN (tumor suppressor gene phosphatase and tensin homologue deleted on chromosome ten) was attenuated upon NGF, but not CCh treatment. This indicates that downregulation of PTEN may help to sustain the phosphorylation of Akt/tuberin by NGF. Collectively, these findings suggest that PP2A and PTEN may be involved in fine tuning the regulation of Akt/tuberin/mTOR/p70S6K in PC12 cells by M₄ mAChR and TrkA, respectively.     

Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells

B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.     

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

Insulin promotes rat retinal neuronal cell survival in a p70S6K-dependent manner

The purpose of this study was to examine the role of the ribosomal protein S6 protein kinase (p70S6K), a protein synthesis regulator, in promoting retinal neuronal cell survival. Differentiated R28 rat retinal neuronal cells were used as an experimental model. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and during the period of experimentation were exposed either to the absence or presence of 10 nm insulin. Insulin treatment induced p70S6K, mTOR, and Akt phosphorylation, effects that were completely prevented by the PI3K inhibitor, LY294002. Insulin-induced phosphorylation of p70S6K and mTOR was prevented by the mTOR inhibitor, rapamycin. Apoptosis, induced by serum deprivation and evaluated by Hoechst staining, was inhibited by insulin treatment in R28 cells, but not in L6 muscle cells. This effect of insulin was also largely prevented by rapamycin. Inhibition of p70S6K activity by exogenous expression of a dominant negative mutant of p70S6K prevented insulin-induced cell survival, whereas, overexpression of wild type p70S6K or expression of a rapamycin resistant form of the kinase enhanced the effect of insulin on survival. Enhanced cell survival under the latter condition was accompanied by increased p70S6K activity and phosphorylation. Rapamycin did not inhibit insulin induced p70S6K phosphorylation and activity in cells transfected with the rapamycin-resistant mutant. Together, these results suggest that p70S6K plays a key role in insulin stimulated retinal neuronal cell survival.     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

Piperlongumine,an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells

The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer.     

EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.     

L-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways

Although amino acids can function as signaling molecules in the regulation of many cellular processes, mechanisms surrounding L-threonine involvement in embryonic stem cell (ESC) functions have not been explored. Thus, we investigated the effect of L-threonine on regulation of mouse (m)ESC self-renewal and related signaling pathways. In L-threonine-depleted mESC culture media mRNA of self-renewal marker genes, [(3)H]thymidine incorporation, expression of c-Myc, Oct4, and cyclins protein was attenuated. In addition, resupplying L-threonine (500 μM) after depletion restores/maintains the mESC proliferation. Disruption of the lipid raft/caveolae microdomain through treatment with methyl-β-cyclodextrin or transfection with caveolin-1 specific small interfering RNA blocked L-threonine-induced proliferation of mESCs. Addition of L-threonine induced phosphorylation of Akt, ERK, p38, JNK/SAPK, and mTOR in a time-dependent manner. This activity was blocked by LY 294002 (PI3K inhibitor), wortmannin (PI3K inhibitor), or an Akt inhibitor. L-threonine-induced activation of mTOR, p70S6K, and 4E-BP1 as well as cyclins and Oct4 were blocked by PD 98059 (ERK inhibitor), SB 203580 (p38 inhibitor) or SP 600125 (JNK inhibitor). Furthermore, L-threonine induced phosphorylation of raptor and rictor binding to mTOR was completely inhibited by 24 h treatment with rapamycin (mTOR inhibitor); however, a 10 min treatment with rapamycin only partially inhibited rictor phosphorylation. L-threonine induced translocation of rictor from the membrane to the cytosol/nuclear, which blocked by pretreatment with rapamycin. In addition, rapamycin blocked L-threonine-induced increases in mRNA expressions of trophoectoderm and mesoderm marker genes and mESC proliferation. In conclusion, L-threonine stimulated ESC G(1)/S transition through lipid raft/caveolae-dependent PI3K/Akt, MAPKs, mTOR, p70S6K, and 4E-BP1 signaling pathways.     

Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts

We have demonstrated that T3 increases the expression of ZAKI-4alpha, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4alpha expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4alpha was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70(S6K), a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Deltap85alpha). Furthermore, the association between thyroid hormone receptor beta1 (TRbeta1) and PI3K-regulatory subunit p85alpha, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70(S6K) cascade by T3 provides a new molecular mechanism for thyroid hormone action.     

Akt2, but not Akt1 or Akt3 mediates pressure-stimulated serum-opsonized latex bead phagocytosis through activating mTOR and p70 S6 kinase

Monocytes and macrophages play critical roles in innate host defense and are sensitive to mechanical stimuli. Tissue pressure is often altered in association with inflammation or infection. Low pressure (20 mmHg), equivalent to normal tissue pressure, increases phagocytosis by primary monocytes and PMA-differentiated THP-1 macrophages, in part by FAK and ERK inhibition and p38 activation. PI-3K is required for macrophage phagocytosis, but whether PI-3K mediates pressure-stimulated phagocytosis is not known. Furthermore, little is known about the role played by the PI-3K downstream Kinases, Akt, and p70 S6 kinase (p70S6K) in modulating macrophage phagocytosis. Thus, we studied the contribution of PI-3K, Akt, and p70S6K to pressure-increased serum-opsonized bead phagocytosis. Pressure-induced p85 PI-3K translocation from cytosolic to membrane fractions and increased Akt activation by 36.1 +/- 12.0% in THP-1 macrophages. LY294002 or Akt inhibitor IV abrogated pressure-stimulated but not basal phagocytosis. Basal Akt activation was inhibited 90% by LY294002 and 70% by Akt inhibitor IV. Each inhibitor prevented Akt activation by pressure. SiRNA targeted to Akt1, Akt2, or Akt3 reduced Akt1, Akt2, and Akt3 expression by 50%, 45%, and 40%, respectively. However, only Akt2SiRNA abrogated the pressure-stimulated phagocytosis without affecting basal. Pressure also activated mTOR and p70S6K. mTORSiRNA and p70S6K inhibition by rapamycin or p70S6KSiRNA blocked pressure-induced, but not basal, phagocytosis. Changes in tissue pressure during inflammation may regulate macrophage phagocytosis by activation of PI-3K, which activates Akt2, mTOR, and p70S6K.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)–p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR–p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR–p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR–p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR–p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR–p70S6K pathway during ischemia. This challenges the accepted notion that mTOR–p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism.     

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.