Science.gov

Sample records for downstream akt activation

  1. Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

    PubMed Central

    McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

  2. Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

    PubMed

    McNamara, Colleen R; Ahuja, Ruchita; Osafo-Addo, Awo D; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

  3. AKT/PKB Signaling: Navigating Downstream

    PubMed Central

    Manning, Brendan D.; Cantley, Lewis C.

    2009-01-01

    The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration. PMID:17604717

  4. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  5. In brain, Axl recruits Grb2 and the p85 regulatory subunit of Pl3 kinase; in vitro mutagenesis defines th requisite binding sites for downstream Akt activation

    PubMed Central

    Weinger, Jason G.; Gohari, Pouyan; Yan, Ying; Backer, Jonathan M.; Varnum, Brian; Shafit-Zagardo, Bridget

    2010-01-01

    Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4– flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways. PMID:18346204

  6. Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways.

    PubMed

    Xia, Wenle; Mullin, Robert J; Keith, Barry R; Liu, Lei-Hua; Ma, Hong; Rusnak, David W; Owens, Gary; Alligood, Krystal J; Spector, Neil L

    2002-09-12

    Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors, as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule, GW572016, potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2, and inhibited activation of Erk1/2 and AKT, downstream effectors of proliferation and cell survival, respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF, often elevated in cancer patients, did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo, where GW572016 treatment inhibited activation of EGFR, erbB2, Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy, or may enhance the anti-tumor activity of chemotherapeutics, since constitutive activation of AKT has been linked to chemo-resistance.

  7. AKT2 is a downstream target of metabotropic glutamate receptor 1 (Grm1).

    PubMed

    Shin, Seung-Shick; Wall, Brian A; Goydos, James S; Chen, Suzie

    2010-02-01

    We reported earlier on the oncogenic properties of Grm1 by demonstrating that stable Grm1-mouse-melanocytic clones proliferate in the absence of growth supplement and anchorage in vitro. In addition, these clones also exhibit aggressive tumorigenic phenotypes in vivo with short latency in tumor formation in both immunodeficient and syngeneic mice. We also detected strong activation of AKT in allograft tumors specifically AKT2 as the predominant isoform involved. In parallel, we assessed several human melanoma biopsy samples and found again that AKT2 was the predominantly activated AKT in these human melanoma biopsies. In cultured stable Grm1-mouse-melanocytic clones, as well as an metabotropic glutamate receptor 1 (Grm1) expressing human melanoma cell line, C8161, stimulation of Grm1 by its agonist led to the activation of AKT, while preincubation with Grm1-antagonist abolished Grm1-agonist-induced AKT activation. In addition, a reduction in tumor volume of Grm1-mouse-melanocytic-allografts was detected in the presence of small interfering AKT2 RNA (siAKT2). Taken together, these results showed that, in addition to the MAPK pathway previously reported being a downstream target of stimulated Grm1, AKT2 is another downstream target in Grm1 mediated melanocyte transformation.

  8. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

    PubMed Central

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C.; Perino, Alessia; Longo, Dario L.; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D.; Backer, Jonathan M.; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  9. Metastasis and AKT activation.

    PubMed

    Qiao, Meng; Sheng, Shijie; Pardee, Arthur B

    2008-10-01

    Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumor's environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3beta causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.

  10. Cyclophilin A as a downstream effector of PI3K/Akt signalling pathway in multiple myeloma cells.

    PubMed

    Lin, Zuo-Lin; Wu, Hsin-Jou; Chen, Jin-An; Lin, Kuo-Chih; Hsu, Jung-Hsin

    2015-12-01

    Cyclophilin A (Cyp A), a member of the peptidyl-prolyl isomerase (PPI) family, may function as a molecular signalling switch. Comparative proteomic studies have identified Cyp A as a potential downstream target of protein kinase B (Akt). This study confirmed that Cyp A is a downstream effector of the phosphatidylinositide 3-kinase (PI3K)/Akt signalling pathway. Cyp A was highly phosphorylated in response to interleukin-6 treatment, which was consistent with the accumulation of phosphorylated Akt, suggesting that Cyp A is a phosphorylation target of Akt and downstream effector of the PI3K/Akt pathway. Cyclosporine A (CsA), a PPI inhibitor, inhibited the growth of multiple myeloma (MM) U266 cells. Moreover, CsA treatment inhibited the activation of the signal transducer and activator of transcription 3 (STAT3) in MM U266 cells. Several Cyp A mutants were generated. Mutants with mutated AKT phosphorylation sites increased the G1 phase arrest in MM U266 cells. The other mutants that mimicked the phosphorylated state of Cyp A decreased the percentage of G1 phase. These results demonstrated that the states of phosphorylation of Cyp A by Akt can influence the progress of the cell cycle in MM U266 cells and that this effect is probably mediated through the Janus-activated kinase 2/STAT3 signalling pathway.

  11. Metastasis and AKT activation.

    PubMed

    Sheng, Shijie; Qiao, Meng; Pardee, Arthur B

    2009-03-01

    Metastasis, responsible for 90% of cancer patient deaths, is an inefficient process because many tumor cells die. The survival of metastatic tumor cells should be considered as a critical therapeutic target. This review provides a new perspective regarding the role of AKT in tumor survival, and the rationale to target AKT in anti-metastasis therapies.

  12. Phosphatidylserine is a critical modulator for Akt activation

    PubMed Central

    Huang, Bill X.; Akbar, Mohammed; Kevala, Karl

    2011-01-01

    Akt activation relies on the binding of Akt to phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the membrane. Here, we demonstrate that Akt activation requires not only PIP3 but also membrane phosphatidylserine (PS). The extent of insulin-like growth factor–induced Akt activation and downstream signaling as well as cell survival under serum starvation conditions positively correlates with plasma membrane PS levels in living cells. PS promotes Akt-PIP3 binding, participates in PIP3-induced Akt interdomain conformational changes for T308 phosphorylation, and causes an open conformation that allows for S473 phosphorylation by mTORC2. PS interacts with specific residues in the pleckstrin homology (PH) and regulatory (RD) domains of Akt. Disruption of PS–Akt interaction by mutation impairs Akt signaling and increases susceptibility to cell death. These data identify a critical function of PS for Akt activation and cell survival, particularly in conditions with limited PIP3 availability. The novel molecular interaction mechanism for Akt activation suggests potential new targets for controlling Akt-dependent cell survival and proliferation. PMID:21402788

  13. Age-associated decrease in contraction-induced activation of downstream targets of Akt/mTor signaling in skeletal muscle.

    PubMed

    Funai, Katsuhiko; Parkington, Jascha D; Carambula, Silvia; Fielding, Roger A

    2006-04-01

    In this study, we investigated the effect of age on the association of eukaryotic initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G), as well as the activity of its binding protein (4E-BP1) and the activity of glycogen synthase kinase-3 (GSK-3) after a single bout of rat hindlimb muscle contractile activity elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Tibialis anterior (TA) and plantaris (Pla) muscles from adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats were collected immediately or 6 h after HFES. eIF4E-eIF4G association was elevated at 6 h of recovery in TA (1.9 +/- 0.2-fold, P < 0.05) and immediately and 6 h after exercise in Pla (2.1 +/- 0.3- and 2.1 +/- 0.7-fold, P < 0.05) in Y rats. No significant increase was observed in O rats. An increase in 4E-BP1 phosphorylation was observed only 6 h after HFES in TA (5.0 +/- 2.0-fold, P < 0.05) in Y rats. Phosphorylation of GSK-3alpha was increased immediately and 6 h after contraction in TA (1.6 +/- 0.3- and 4.1 +/- 0.8-fold, P < 0.05) and Pla (1.7 +/- 0.2- and 2.1 +/- 0.4-fold, P < 0.05) in Y rats and remained unaffected in O rats. Phosphorylation of GSK-3beta was observed only immediately after HFES in TA (1.5 +/- 0.2-fold, P < 0.05) in Y rats. Overall, eIF4E-eIF4G association and phosphorylation of 4E-BP1 and GSK-3 are increased after HFES in adult, but not in aged, animals. These observations suggest that the anabolic response to muscle stimulation is attenuated with aging and may contribute to the limited capacity of hypertrophy in aged animals.

  14. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

    SciTech Connect

    Wang, Yelin; Hu, Chen; Cheng, Jun; Chen, Binquan; Ke, Qinghong; Lv, Zhen; Wu, Jian; Zhou, Yanfeng

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  15. AKT1 and AKT2 isoforms play distinct roles during breast cancer progression through the regulation of specific downstream proteins

    PubMed Central

    Riggio, Marina; Perrone, María C.; Polo, María L.; Rodriguez, María J.; May, María; Abba, Martín; Lanari, Claudia; Novaro, Virginia

    2017-01-01

    The purpose of this study was to elucidate the mechanisms associated with the specific effects of AKT1 and AKT2 isoforms in breast cancer progression. We modulated the abundance of specific AKT isoforms in IBH-6 and T47D human breast cancer cell lines and showed that AKT1 promoted cell proliferation, through S6 and cyclin D1 upregulation, but it inhibited cell migration and invasion through β1-integrin and focal adhesion kinase (FAK) downregulation. In contrast, AKT2 promoted cell migration and invasion through F-actin and vimentin induction. Thus, while overexpression of AKT1 promoted local tumor growth, downregulation of AKT1 or overexpression of AKT2 promoted peritumoral invasion and lung metastasis. Furthermore, we evaluated The Cancer Genome Atlas (TCGA) dataset for invasive breast carcinomas and found that increased AKT2 but not AKT1 mRNA levels correlated with a worse clinical outcome. We conclude that AKT isoforms play specific roles in different steps of breast cancer progression, with AKT1 involved in the local tumor growth and AKT2 involved in the distant tumor dissemination, having AKT2 a poorer prognostic value and consequently being a worthwhile target for therapy. PMID:28287129

  16. Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

    PubMed Central

    Rudelius, Martina; Pittaluga, Stefania; Nishizuka, Satoshi; Pham, Trinh H.-T.; Fend, Falko; Jaffe, Elaine S.; Quintanilla-Martinez, Leticia; Raffeld, Mark

    2006-01-01

    To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt and multiple downstream targets in primary MCL cases and cell lines. Akt was phosphorylated in 12 of 12 aggressive blastoid MCL variants and in 4 of 4 MCL cell lines. In contrast, phosphorylated Akt was present in only 5 of 16 typical MCL, 3 at comparable levels to the blastoid cases, and 2 at low levels. The presence of p-Akt was accompanied by the phosphorylation of p27kip1, FRKHL-1, MDM2, Bad, mTOR, and p70S6K. Inhibition of the PI3K/Akt pathway in the MCL cell lines abrogated or reduced the phosphorylation of Akt, p27kip1, FRKHL-1, MDM2, Bad, mTOR, GSK-3β, IκB, and led to cell-cycle arrest and apoptosis. Six MCL cases (5 with activated Akt and 1 with inactive Akt) and 3 of 4 cell lines showed loss of PTEN expression. PIK3CA mutations were not detected. We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL and preferentially occurs in blastoid variants. One possible mechanism of activation is loss of PTEN expression. These data suggest that PI3K/Akt inhibitors may be effective in the treatment of Akt-activated MCL. PMID:16645163

  17. Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Pise-Masison, Cynthia A; Radonovich, Michael F; Park, Hyeon Ung; Brady, John N

    2005-10-06

    AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.

  18. Genetic dissection reveals that Akt is the critical kinase downstream of LRRK2 to phosphorylate and inhibit FOXO1, and promotes neuron survival.

    PubMed

    Chuang, Chia-Lung; Lu, Yu-Ning; Wang, Hung-Cheng; Chang, Hui-Yun

    2014-11-01

    Leucine-rich repeat kinase 2 (LRRK2) is a complex kinase and mutations in LRRK2 are perhaps the most common genetic cause of Parkinson's disease (PD). However, the identification of the normal physiological function of LRRK2 remains elusive. Here, we show that LRRK2 protects neurons against apoptosis induced by the Drosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinase of LRRK2 that phosphorylates and inhibits FOXO1, and thereby promotes survival. Like human LRRK2, Drosophila lrrk also promotes neuron survival; lrrk loss-of-function mutant displays reduced cell numbers, which can be rescued by LRRK2 expression. Importantly, LRRK2 G2019S and LRRK2 R1441C mutants impair the ability of LRRK2 to activate Akt, and fail to prevent apoptotic death. Ectopic expression of a constitutive active form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can protect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and then inhibits FOXO1. These results might indicate that a LRRK-Akt therapeutic pathway to promote neuron survival and to prevent neurodegeneration in Parkinson's disease. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Cross Talk between the Akt and p38α Pathways in Macrophages Downstream of Toll-Like Receptor Signaling

    PubMed Central

    McGuire, Victoria A.; Gray, Alexander; Monk, Claire E.; Santos, Susana G.; Lee, Keunwook; Aubareda, Anna; Crowe, Jonathan; Ronkina, Natalia; Schwermann, Jessica; Batty, Ian H.; Leslie, Nick R.; Dean, Jonathan L. E.; O'Keefe, Stephen J.; Boothby, Mark; Gaestel, Matthias

    2013-01-01

    The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)–Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages. PMID:23979601

  20. Site Specific Activation of AKT Protects Cells from Death Induced by Glucose Deprivation

    PubMed Central

    Gao, Meng; Liang, Jiyong; Lu, Yiling; Guo, Huifang; German, Peter; Bai, Shanshan; Jonasch, Eric; Yang, Xingsheng; Mills, Gordon B.; Ding, Zhiyong

    2013-01-01

    The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. PMID:23396361

  1. AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells.

    PubMed

    Abdelhamed, Sherif; Ogura, Keisuke; Yokoyama, Satoru; Saiki, Ikuo; Hayakawa, Yoshihiro

    2016-01-01

    While cancer development and progression can be controlled by cytotoxic T cells, it is also known that tumor-specific CD8(+)T cells become functionally impaired by acquiring a group of inhibitory receptors known as immune checkpoints. Amongst those, programmed death-1 (PD-1) is one of the most recognized negative regulators of T cell function. In non-small lung cancers (NSCLCs), the aberrant activation of epidermal growth factor receptor (EGFR) is known to induce PD-L1 expression and further the treatment with gefitinib, a tyrosine kinase inhibitor (TKI) for EGFR, decrease the expression of PD-L1 on NSCLC. Given the acquired resistance to gefitinib treatment frequently observed by developing secondary-site mutations limiting its efficacy, it is important to understand the downstream mechanism of activated-EGFR signaling for regulating PD-L1 in NSCLC. In this study, we demonstrated that AKT-STAT3 pathway could be a potential target for regulating the surface expression of PD-L1 on NSCLCs with aberrant EGFR activity and, further, the inhibition of AKT or STAT3 activity could down-regulate the expression of PD-L1 even in gefitinib-resistant NSCLCs. These results highlight an importance of AKT-STAT3 pathway as a promising target for potentiating anti-tumor immune responses by regulating PD-L1 expression on cancer cells with aberrant EGFR activity.

  2. Akt phosphorylates and regulates the osteogenic activity of Osterix.

    PubMed

    Choi, You Hee; Jeong, Hyung Min; Jin, Yun-Hye; Li, Hongyan; Yeo, Chang-Yeol; Lee, Kwang-Youl

    2011-08-05

    Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.

  3. Akt is required for Stat5 activation and mammary differentiation

    PubMed Central

    2010-01-01

    Introduction The Akt pathway plays a central role in regulating cell survival, proliferation and metabolism, and is one of the most commonly activated pathways in human cancer. A role for Akt in epithelial differentiation, however, has not been established. We previously reported that mice lacking Akt1, but not Akt2, exhibit a pronounced metabolic defect during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Despite this metabolic defect, however, both Akt1-deficient and Akt2-deficient mice exhibit normal mammary epithelial differentiation and Stat5 activation. Methods In light of the overlapping functions of Akt family members, we considered the possibility that Akt may play an essential role in regulating mammary epithelial development that is not evident in Akt1-deficient mice due to compensation by other Akt isoforms. To address this possibility, we interbred mice bearing targeted deletions in Akt1 and Akt2 and determined the effect on mammary differentiation during pregnancy and lactation. Results Deletion of one allele of Akt2 in Akt1-deficient mice resulted in a severe defect in Stat5 activation during late pregnancy that was accompanied by a global failure of terminal mammary epithelial cell differentiation, as manifested by the near-complete loss in production of the three principal components of milk: lactose, lipid, and milk proteins. This defect was due, in part, to a failure of pregnant Akt1-/-;Akt2+/- mice to upregulate the positive regulator of Prlr-Jak-Stat5 signaling, Id2, or to downregulate the negative regulators of Prlr-Jak-Stat5 signaling, caveolin-1 and Socs2. Conclusions Our findings demonstrate an unexpected requirement for Akt in Prlr-Jak-Stat5 signaling and establish Akt as an essential central regulator of mammary epithelial differentiation and lactation. PMID:20849614

  4. Turbulence decay downstream of an active grid

    NASA Astrophysics Data System (ADS)

    Bewley, Gregory; Bodenschatz, Eberhard

    2015-11-01

    A grid in a wind tunnel stirs up turbulence that has a certain large-scale structure. The moving parts in a so-called ``active grid'' can be programmed to produce different structures. We use a special active grid in which each of 129 paddles on the grid has its own position-controlled servomotor that can move independently of the others. We observe among other things that the anisotropy in the amplitude of the velocity fluctuations and in the correlation lengths can be set and varied with an algorithm that oscillates the paddles in a specified way. The variation in the anisotropies that we observe can be explained by our earlier analysis of anisotropic ``soccer ball'' turbulence (Bewley, Chang and Bodenschatz 2012, Phys. Fluids). We define the influence of this variation in structure on the downstream evolution of the turbulence. with Eberhard Bodenschatz and others.

  5. Akt and mTOR in B Cell Activation and Differentiation.

    PubMed

    Limon, Jose J; Fruman, David A

    2012-01-01

    Activation of phosphoinositide 3-kinase (PI3K) is required for B cell proliferation and survival. PI3K signaling also controls key aspects of B cell differentiation. Upon engagement of the B cell receptor (BCR), PI3K activation promotes Ca(2+) mobilization and activation of NFκB-dependent transcription, events which are essential for B cell proliferation. PI3K also initiates a distinct signaling pathway involving the Akt and mTOR serine/threonine kinases. It has been generally assumed that activation of Akt and mTOR downstream of PI3K is essential for B cell function. However, Akt and mTOR have complex roles in B cell fate decisions and suppression of this pathway can enhance certain B cell responses while repressing others. In this review we will discuss genetic and pharmacological studies of Akt and mTOR function in normal B cells, and in malignancies of B cell origin.

  6. Photoreceptor Neuroprotection: Regulation of Akt Activation Through Serine/Threonine Phosphatases, PHLPP and PHLPPL.

    PubMed

    Rajala, Raju V S; Kanan, Yogita; Anderson, Robert E

    2016-01-01

    Serine/threonine kinase Akt is a downstream effector of insulin receptor/PI3K pathway that is involved in many processes, including providing neuroprotection to stressed rod photoreceptor cells. Akt signaling is known to be regulated by the serine/threonine phosphatases, PHLPP (PH domain and leucine rich repeat protein phosphatase) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like). We previously reported that both phosphatases are expressed in the retina, as well as in photoreceptor cells. In this study, we examined the PHLPP and PHLPPL phosphatase activities towards non-physiological and physiological substrates. Our results suggest that PHLPP was more active than PHLPPL towards non-physiological substrates, whereas both PHLPP and PHLPP dephosphorylated the physiological substrates of Akt1 and Akt3 with similar efficiencies. Our results also suggest that knockdown of PHLPPL alone does not increase Akt phosphorylation, due to a compensatory increase of PHLPP, which results in the dephosphorylation of Akt. Therefore, PHLPP and PHLPPL regulate Akt activation together when both phosphatases are expressed.

  7. Chloroquine stimulates glucose uptake and glycogen synthase in muscle cells through activation of Akt.

    PubMed

    Halaby, Marie-Jo; Kastein, Brandon K; Yang, Da-Qing

    2013-06-14

    Chloroquine is a pharmaceutical agent that has been widely used to treat patients with malaria. Chloroquine has also been reported to have hypoglycemic effects on humans and animal models of diabetes. Despite many previous studies, the mechanism responsible for its hypoglycemic effect is still unclear. Chloroquine was recently reported to be an activator of ATM, the protein deficient in the Ataxia-telagiectasia (A-T) disease. Since ATM is also known as an insulin responsive protein that mediates Akt activation, we tested the effect of chloroquine on the activity of Akt and its downstream targets. In L6 muscle cells treated with insulin and chloroquine, the phosphorylation of Akt and glucose uptake were dramatically increased compared to cells treated with insulin alone, suggesting that chloroquine is a potent activator of Akt and glucose uptake in these cells. We also found that the reduction of insulin-mediated Akt activity in muscle tissues of insulin resistant rats was partially reversed by chloroquine treatment. Moreover, insulin-mediated phosphorylation of glycogen synthase kinase-3β in L6 cells was greatly enhanced by chloroquine. A substantial decrease in phosphorylation of glycogen synthase was also observed in chloroquine-treated L6 cells, indicating enhanced activity of glycogen synthase. Taken together, our results not only show that chloroquine is a novel activator of Akt that stimulates glucose uptake and glycogen synthase, but also validate chloroquine as a potential therapeutic agent for patients with type 2 diabetes mellitus. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. The effect to IL-3Ralpha, downstream PI3k/Akt signaling of all-trans retinoic acid and arsenic trioxide in NB4 cells.

    PubMed

    Chen, Ping; Wu, Juan-Ying; Huang, Hui-Fang; Chen, Yuan-Zhong

    2014-04-01

    All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are the classic drugs used for induction therapy of acute promyelocytic leukemia (APL). IL-3Ralpha (CD123) is a specific marker of acute myeloid leukemia stem cells (AML-LSCs). The over-expression of IL-3Ralpha in patients with AML is related to high white blood cells counts, high percentages of blast cells, and poor prognosis. Moreover, in some studies, IL-3Ralpha has been considered a new detection marker of minimal residual disease in the bone marrow from patients with APL. In contrast to ATRA, As2O3 reduces both mRNA and protein expression of IL-3Ralpha and inhibits the activity of PI3K/Akt after 24 h, 48 h, and 72 h of exposure. Furthermore, NB4 cells adhered to the human stroma cell line HS-5 cells were used as an in vitro model of APL cells in the bone marrow microenvironment. Our results demonstrate that adhesion to HS-5 cells up-regulated IL-3Ralpha protein expression and activated the downstream PI3K/Akt signaling pathway in NB4 cells. Compared with ATRA, As2O3 more potently inhibits proliferation of NB4 cells adhered to stroma cells.

  9. CKIP-1 regulates macrophage proliferation by inhibiting TRAF6-mediated Akt activation

    PubMed Central

    Zhang, Luo; Wang, Yiwu; Xiao, Fengjun; Wang, Shaoxia; Xing, Guichun; Li, Yang; Yin, Xiushan; Lu, Kefeng; Wei, Rongfei; Fan, Jiao; Chen, Yuhan; Li, Tao; Xie, Ping; Yuan, Lin; Song, Lei; Ma, Lanzhi; Ding, Lujing; He, Fuchu; Zhang, Lingqiang

    2014-01-01

    Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1−/− mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation. PMID:24777252

  10. Progestins Activate the AKT Pathway in Leiomyoma Cells and Promote Survival

    PubMed Central

    Hoekstra, Anna V.; Sefton, Elizabeth C.; Berry, Emily; Lu, Zhenxiao; Hardt, Jennifer; Marsh, Erica; Yin, Ping; Clardy, Jon; Chakravarti, Debabrata; Bulun, Serdar; Kim, J. Julie

    2009-01-01

    Context: Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear. Objective: In this study we investigated the activation of the AKT pathway and its downstream effectors, glycogen synthase kinase-3b and Forkhead box O (FOXO)-1 by progesterone as a mechanism of proliferation and survival of leiomyoma cells. Inhibitors of the AKT pathway were used to demonstrate the role of phosphatidylinositol 3-kinase, AKT, and FOXO1 in contributing to cell proliferation and apoptosis. Results: Treatment of leiomyoma cells with R5020 over a period of 72 h resulted in higher cell numbers compared with untreated cells. When cells were treated with 100 nm R5020 for 1 and 24 h, the levels of phospho(Ser 473)-AKT increased. This increase was inhibited when cells were cotreated with RU486. Treatment of leiomyoma cells with a phosphatidylinositol 3-kinase inhibitor, LY294 dramatically decreased levels of phospho(Ser 473)-AKT, despite R5020 treatment. In addition to increased phospho(Ser 473)-AKT levels, R5020 treatment resulted in an increase in phospho(Ser 256)-FOXO1 and phosphoglycogen synthase kinase-3b. Inhibition of AKT using API-59 decreased proliferation and cell viability even in the presence of R5020. Higher concentrations of API-59-induced apoptosis of leiomyoma cells, even in the presence of R5020. Psammaplysene A increased nuclear FOXO1 levels and did not affect cell proliferation but induced apoptosis of leiomyoma cells. Conclusions: The progestin, R5020, can rapidly activate the AKT pathway. Inhibition of the AKT pathway inhibits cell proliferation and promotes apoptosis of leiomyoma cells. PMID:19240153

  11. Unique Effects of Acute Aripiprazole Treatment on the Dopamine D2 Receptor Downstream cAMP-PKA and Akt-GSK3β Signalling Pathways in Rats

    PubMed Central

    Pan, Bo; Chen, Jiezhong; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2015-01-01

    Aripiprazole is a wide-used antipsychotic drug with therapeutic effects on both positive and negative symptoms of schizophrenia, and reduced side-effects. Although aripiprazole was developed as a dopamine D2 receptor (D2R) partial agonist, all other D2R partial agonists that aimed to mimic aripiprazole failed to exert therapeutic effects in clinic. The present in vivo study aimed to investigate the effects of aripiprazole on the D2R downstream cAMP-PKA and Akt-GSK3β signalling pathways in comparison with a D2R antagonist – haloperidol and a D2R partial agonist – bifeprunox. Rats were injected once with aripiprazole (0.75mg/kg, i.p.), bifeprunox (0.8mg/kg, i.p.), haloperidol (0.1mg/kg, i.p.) or vehicle. Five brain regions – the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), ventral tegmental area (VTA) and substantia nigra (SN) were collected. The protein levels of PKA, Akt and GSK3β were measured by Western Blotting; the cAMP levels were examined by ELISA tests. The results showed that aripiprazole presented similar acute effects on PKA expression to haloperidol, but not bifeprunox, in the CPU and VTA. Additionally, aripiprazole was able to increase the phosphorylation of GSK3β in the PFC, NAc, CPu and SN, respectively, which cannot be achieved by bifeprunox and haloperidol. These results suggested that acute treatment of aripiprazole had differential effects on the cAMP-PKA and Akt-GSK3β signalling pathways from haloperidol and bifeprunox in these brain areas. This study further indicated that, by comparison with bifeprunox, the unique pharmacological profile of aripiprazole may be attributed to the relatively lower intrinsic activity at D2R. PMID:26162083

  12. Unique Effects of Acute Aripiprazole Treatment on the Dopamine D2 Receptor Downstream cAMP-PKA and Akt-GSK3β Signalling Pathways in Rats.

    PubMed

    Pan, Bo; Chen, Jiezhong; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2015-01-01

    Aripiprazole is a wide-used antipsychotic drug with therapeutic effects on both positive and negative symptoms of schizophrenia, and reduced side-effects. Although aripiprazole was developed as a dopamine D2 receptor (D2R) partial agonist, all other D2R partial agonists that aimed to mimic aripiprazole failed to exert therapeutic effects in clinic. The present in vivo study aimed to investigate the effects of aripiprazole on the D2R downstream cAMP-PKA and Akt-GSK3β signalling pathways in comparison with a D2R antagonist--haloperidol and a D2R partial agonist--bifeprunox. Rats were injected once with aripiprazole (0.75 mg/kg, i.p.), bifeprunox (0.8 mg/kg, i.p.), haloperidol (0.1 mg/kg, i.p.) or vehicle. Five brain regions--the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), ventral tegmental area (VTA) and substantia nigra (SN) were collected. The protein levels of PKA, Akt and GSK3β were measured by Western Blotting; the cAMP levels were examined by ELISA tests. The results showed that aripiprazole presented similar acute effects on PKA expression to haloperidol, but not bifeprunox, in the CPU and VTA. Additionally, aripiprazole was able to increase the phosphorylation of GSK3β in the PFC, NAc, CPu and SN, respectively, which cannot be achieved by bifeprunox and haloperidol. These results suggested that acute treatment of aripiprazole had differential effects on the cAMP-PKA and Akt-GSK3β signalling pathways from haloperidol and bifeprunox in these brain areas. This study further indicated that, by comparison with bifeprunox, the unique pharmacological profile of aripiprazole may be attributed to the relatively lower intrinsic activity at D2R.

  13. FANCI is a negative regulator of Akt activation.

    PubMed

    Zhang, Xiaoshan; Lu, Xiaoyan; Akhter, Shamima; Georgescu, Maria-Magdalena; Legerski, Randy J

    2016-01-01

    Akt is a critical mediator of the oncogenic PI3K pathway, and its activation is regulated by kinases and phosphatases acting in opposition. We report here the existence of a novel protein complex that is composed minimally of Akt, PHLPP1, PHLPP2, FANCI, FANCD2, USP1 and UAF1. Our studies show that depletion of FANCI, but not FANCD2 or USP1, results in increased phosphorylation and activation of Akt. This activation is due to a reduction in the interaction between PHLPP1 and Akt in the absence of FANCI. In response to DNA damage or growth factor treatment, the interactions between Akt, PHLPP1 and FANCI are reduced consistent with the known phosphorylation of Akt in response to these stimuli. Furthermore, depletion of FANCI results in reduced apoptosis after DNA damage in accord with its role as a negative regular of Akt. Our findings describe an unexpected function for FANCI in the regulation of Akt and define a previously unrecognized intersection between the PI3K-Akt and FA pathways.

  14. Effects of the Activation of Three Major Hepatic Akt Substrates on Glucose Metabolism in Male Mice.

    PubMed

    Sakai, Gota; Inoue, Ikuo; Suzuki, Tokuko; Sumita, Takashi; Inukai, Kouichi; Katayama, Shigehiro; Awata, Takuya; Yamada, Tetsuya; Asano, Tomoichiro; Katagiri, Hideki; Noda, Mitsuhiko; Shimada, Akira; Ono, Hiraku

    2017-08-01

    Insulin suppresses glucose output from the liver via Akt activation; however, which substrate of Akt plays the major role in transducing this effect is unclear. We tested the postnatal expression of Akt-unresponsive, constitutively active mutants of three major Akt substrates widely considered to regulate glucose metabolism [i.e., FoxO1, PGC1α, and glycogen synthase kinase-3β (GSK3β)] using adenoviral gene delivery to the mouse liver. We performed physiological hyperinsulinemic-euglycemic clamp studies using these mice under awake and nonrestrained conditions with blood sampling via an arterial catheter. Hepatic expression of constitutively active FoxO1 induced significant hepatic and systemic insulin resistance. However, neither the expression of constitutively active PGC1α nor that of GSK3β significantly changed insulin sensitivity. Simultaneous expression of all three mutants together induced no further insulin resistance compared with that of the FoxO1 mutant. The glycogen content in the liver was significantly reduced by constitutively active GSK3β expression. In cultured hepatocytes, constitutively active PGC1α induced markedly stronger transcriptional enhancement of gluconeogenic key enzymes than did constitutively active FoxO1. From these results, we conclude that FoxO1 has the most prominent role in transducing insulin's effect downstream from Akt to suppress hepatic glucose output, involving mechanisms independent of the transcriptional regulation of key gluconeogenic enzymes. Copyright © 2017 Endocrine Society.

  15. Akt phosphorylates Tal1 oncoprotein and inhibits its repressor activity.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-06-01

    The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

  16. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.

    PubMed

    Chen, Jiun-Han; Hsiao, George; Lee, An-Rong; Wu, Chin-Chen; Yen, Mao-Hsiung

    2004-04-01

    Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.

  17. Activation and Membrane Binding of Retinal Protein Kinase Bα/Akt1 is Regulated through Light-Dependent Generation of Phosphoinositides

    PubMed Central

    Li, Guiyuan; Rajala, Ammaji; Wiechmann, Allan F.; Anderson, Robert E.; Rajala, Raju V.S.

    2008-01-01

    Akt is a phospholipid-binding protein and the downstream effector of the phosphoinositide 3-kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K-dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor-specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/Akt1-PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light-dependent manner. This study suggests that light-dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light-dependent generation of phosphoinositides. PMID:18823366

  18. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2009-11-01

    construct from the top, Fig. 1), proved to be incompatible with Akt1 activity. The prenylation signal derived from K- Ras rendered Akt1 catalytically...with either cyclo- dextrin (CD) or water-soluble cholesterol ( Chol ) or with CD followed by cholesterol treatment (CD Chol ). Cells incubated in serum

  19. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2009-07-01

    variant (third construct from the top, Fig. 1), proved to be incompatible with Akt1 activity. The prenylation signal derived from K- Ras rendered Akt1...water-soluble cholesterol ( Chol ) or with CD followed by cholesterol treatment (CD Chol ). Cells incubated in serum-free medium served as controls

  20. Activation of Akt predicts poor outcome in neuroblastoma.

    PubMed

    Opel, Daniela; Poremba, Christopher; Simon, Thorsten; Debatin, Klaus-Michael; Fulda, Simone

    2007-01-15

    Whereas aberrant activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, a key survival cascade, has previously been linked to poor prognosis in several human malignancies, its prognostic effect in neuroblastoma has not yet been explored. We therefore investigated the phosphorylation status of Akt, S6 ribosomal protein as target of mammalian target of rapamycin, and extracellular signal-regulated kinase (ERK) in 116 primary neuroblastoma samples by tissue microarray and its correlation with established prognostic markers and survival outcome. Here, we provide for the first time evidence that phosphorylation of Akt at serine 473 (S473) and/or threonine 308 (T308), S6 ribosomal protein, and ERK frequently occurs in primary neuroblastoma. Importantly, we identified Akt activation as a novel prognostic indicator of decreased event-free or overall survival in neuroblastoma, whereas phosphorylation of S6 ribosomal protein or ERK had no prognostic effect. In addition, Akt activation correlated with variables of aggressive disease, including MYCN amplification, 1p36 aberrations, advanced disease stage, age at diagnosis, and unfavorable histology. Monitoring Akt at T308 or both phosphorylation sites improved the prognostic significance of Akt activation in neuroblastoma specimens compared with S473 phosphorylation. Parallel experiments in neuroblastoma cell lines revealed that activation of Akt by insulin-like growth factor (IGF)-I significantly inhibited tumor necrosis factor-related apoptosis-inducing ligand- or chemotherapy-induced apoptosis in a PI3K-dependent manner because the PI3K inhibitor LY294002 completely reversed the IGF-I-mediated protection of neuroblastoma cells from apoptosis. By showing that activation of Akt correlates with poor prognosis in primary neuroblastoma in vivo and with apoptosis resistance in vitro, our findings indicate that Akt presents a clinically relevant target in neuroblastoma that warrants further investigation.

  1. Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

    PubMed Central

    Raufman, Jean-Pierre; Shant, Jasleen; Guo, Chang Yue; Roy, Sanjit; Cheng, Kunrong

    2010-01-01

    Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-α-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser473) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser21/9) and BAD (Ser136), and nuclear translocation (activation) of NF-κB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation. PMID:18064605

  2. Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling.

    PubMed

    Min, Young Kyu; Lee, Jong Eun; Chung, Kwang Chul

    2007-01-15

    Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.

  3. Somatic Activation of AKT3 Causes Hemispheric Developmental Brain Malformations

    PubMed Central

    Poduri, Annapurna; Evrony, Gilad D.; Cai, Xuyu; Elhosary, Princess Christina; Beroukhim, Rameen; Lehtinen, Maria K.; Hills, L. Benjamin; Heinzen, Erin L.; Hill, Anthony; Hill, R. Sean; Barry, Brenda J.; Bourgeois, Blaise F.D.; Riviello, James J.; Barkovich, A. James; Black, Peter M.; Ligon, Keith L.; Walsh, Christopher A.

    2012-01-01

    Summary Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that 2/8 HMG samples showed trisomy of chromosome 1q, encompassing many genes, including AKT3, which is known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patient’s blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralogue in brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to brain could represent an important cause of complex neurogenetic disease. PMID:22500628

  4. Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

    PubMed Central

    Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

    2013-01-01

    Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3

  5. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival.

    PubMed

    Bozulic, Lana; Surucu, Banu; Hynx, Debby; Hemmings, Brian A

    2008-04-25

    Protein kinase B (PKB/Akt) is a well-established regulator of several essential cellular processes. Here, we report a route by which activated PKB promotes survival in response to DNA insults in vivo. PKB activation following DNA damage requires 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localizes in the nucleus of gamma-irradiated cells adjacent to DNA double-strand breaks, where it colocalizes and interacts with DNA-PK. Levels of active PKB inversely correlate with DNA damage-induced apoptosis. A significant portion of p53- and DNA damage-regulated genes are misregulated in cells lacking PKBalpha. PKBalpha knockout mice show impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single-dose whole-body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a prosurvival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKBalpha isoform.

  6. Multiple host kinases contribute to Akt activation during Salmonella infection.

    PubMed

    Roppenser, Bernhard; Kwon, Hyunwoo; Canadien, Veronica; Xu, Risheng; Devreotes, Peter N; Grinstein, Sergio; Brumell, John H

    2013-01-01

    SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  7. Modulation in Activation and Expression of PTEN, Akt1, and PDK1: Further Evidence Demonstrating Altered Phosphoinositide 3-kinase Signaling in Postmortem Brain of Suicide Subjects

    PubMed Central

    Dwivedi, Yogesh; Rizavi, Hooriyah S.; Zhang, Hui; Roberts, Rosalinda C.; Conley, Robert R.; Pandey, Ghanshyam N.

    2010-01-01

    Background Phosphoinositide 3-kinase (PI 3-K) signaling plays a crucial role in neuronal growth and plasticity. Recently, we demonstrated that suicide brain is associated with decreased activation and expression of selective catalytic and regulatory subunits of PI 3-K. The present investigation examined the regulation and functional significance of compromised PI 3-K in suicide brain at the level of upstream phosphatase and tensin homolog on chromosome ten (PTEN) and downstream substrates 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. Method mRNA expression of Akt1, Akt3, PTEN, and PDK1 by competitive RT-PCR; protein expression of Akt1, Akt3, PTEN, PDK1, phosphorylated-Akt1 (Ser473), phosphorylated-Akt1(Thr308), phosphorylated-PDK1, and phosphorylated-PTEN by Western blot; and catalytic activities of Akt1, Akt3, and PDK1 by enzymatic assays were determined in prefrontal cortex (PFC) and hippocampus obtained from suicide subjects and nonpsychiatric controls. Results No significant changes in the expression of Akt1 or Akt3 were observed; however, catalytic activity of Akt1, but not of Akt3, was decreased in PFC and hippocampus of suicide subjects, which was associated with decreased phosphorylation of Akt1 at Ser473 and Thr308. The catalytic activity of PDK1 and the level of phosphorylated-PDK1 were also decreased in both brain areas without any change in expression levels of PDK1. On the other hand, mRNA and protein expression of PTEN was increased, whereas the level of phosphorylated-PTEN was decreased. Conclusion Our study demonstrates abnormalities in PI 3-K signaling at several levels in brain of suicide subjects and suggests the possible involvement of aberrant PI 3-K/Akt signaling in the pathogenic mechanisms of suicide. PMID:20163786

  8. Direct Regulation of Osteocytic Connexin 43 Hemichannels through AKT Kinase Activated by Mechanical Stimulation*

    PubMed Central

    Batra, Nidhi; Riquelme, Manuel A.; Burra, Sirisha; Kar, Rekha; Gu, Sumin; Jiang, Jean X.

    2014-01-01

    Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone. PMID:24563481

  9. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    PubMed

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.

  10. Moderate exercise promotes human RBC-NOS activity, NO production and deformability through Akt kinase pathway.

    PubMed

    Suhr, Frank; Brenig, Julian; Müller, Rebecca; Behrens, Hilke; Bloch, Wilhelm; Grau, Marijke

    2012-01-01

    Nitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs. Fifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer(1177) was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr(495) were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability. This human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.

  11. Physical association of PDK1 with AKT1 is sufficient for pathway activation independent of membrane localization and phosphatidylinositol 3 kinase.

    PubMed

    Ding, Zhiyong; Liang, Jiyong; Li, Jin; Lu, Yiling; Ariyaratna, Vathsala; Lu, Zhimin; Davies, Michael A; Westwick, John K; Mills, Gordon B

    2010-03-26

    Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.

  12. In vitro and in vivo activity of novel small-molecule inhibitors targeting the pleckstrin homology domain of protein kinase B/AKT.

    PubMed

    Moses, Sylvestor A; Ali, M Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A Geoffrey; Zhang, Shuxing; Mash, Eugene A; Powis, Garth; Meuillet, Emmanuelle J

    2009-06-15

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

  13. Stat5 Promotes Survival of Mammary Epithelial Cells through Transcriptional Activation of a Distinct Promoter in Akt1▿

    PubMed Central

    Creamer, Bradley A.; Sakamoto, Kazuhito; Schmidt, Jeffrey W.; Triplett, Aleata A.; Moriggl, Richard; Wagner, Kay-Uwe

    2010-01-01

    The signal transducer and activator of transcription 5 (Stat5) plays a pivotal role in the proliferation, secretory differentiation, and survival of mammary epithelial cells. However, there is little information about Stat5 target genes that facilitate these biological processes. We provide here experimental evidence that the prolactin-mediated phosphorylation of Stat5 regulates the transcriptional activation of the Akt1 gene. Stat5 binds to consensus sequences within the Akt1 locus in a growth factor-dependent manner to initiate transcription of a unique Akt1 mRNA from a distinct promoter, which is only active in the mammary gland. Elevating the levels of active Akt1 restores the expression of cyclin D1 and proliferation of Jak2-deficient mammary epithelial cells, which provides evidence that Akt1 acts downstream of Jak/Stat signaling. The ligand-inducible expression of Stat5 in transgenic females mediates a sustained upregulation of Akt1 in mammary epithelial cells during the onset of postlactational involution. Stat5-expressing mammary glands exhibit a delay in involution despite induction of proapoptotic signaling events. Collectively, the results of the present study elucidate an underlying mechanism by which active Stat5 mediates evasion from apoptosis and self-sufficiency in growth signals. PMID:20385773

  14. Quantitative network signal combinations downstream of TCR activation can predict IL-2 production response.

    PubMed

    Kemp, Melissa L; Wille, Lucia; Lewis, Christina L; Nicholson, Lindsay B; Lauffenburger, Douglas A

    2007-04-15

    Proximal signaling events activated by TCR-peptide/MHC (TCR-pMHC) binding have been the focus of intense ongoing study, but understanding how the consequent downstream signaling networks integrate to govern ultimate avidity-appropriate TCR-pMHC T cell responses remains a crucial next challenge. We hypothesized that a quantitative combination of key downstream network signals across multiple pathways must encode the information generated by TCR activation, providing the basis for a quantitative model capable of interpreting and predicting T cell functional responses. To this end, we measured 11 protein nodes across six downstream pathways, along five time points from 10 min to 4 h, in a 1B6 T cell hybridoma stimulated by a set of three myelin proteolipid protein 139-151 altered peptide ligands. A multivariate regression model generated from this data compendium successfully comprehends the various IL-2 production responses and moreover successfully predicts a priori the response to an additional peptide treatment, demonstrating that TCR binding information is quantitatively encoded in the downstream network. Individual node and/or time point measurements less effectively accounted for the IL-2 responses, indicating that signals must be integrated dynamically across multiple pathways to adequately represent the encoded TCR signaling information. Of further importance, the model also successfully predicted a priori direct experimental tests of the effects of individual and combined inhibitors of the MEK/ERK and PI3K/Akt pathways on this T cell response. Together, our findings show how multipathway network signals downstream of TCR activation quantitatively integrate to translate pMHC stimuli into functional cell responses.

  15. Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury

    PubMed Central

    Hao, Jia; Ahn, Hee-Yul

    2012-01-01

    Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3β, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3β inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3β. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC. PMID:23000580

  16. Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury.

    PubMed

    Kim, Chan-Hyung; Hao, Jia; Ahn, Hee-Yul; Kim, Si Wook

    2012-09-01

    Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase(PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved postischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3β, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3β inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3(+)), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3β. Furthermore, SIC was abrogated by wortmannin and Gd3(+). In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3(+) pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.

  17. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  18. Akt1 Signaling Regulates Integrin Activation, Matrix Recognition, and Fibronectin Assembly*S

    PubMed Central

    Somanath, Payaningal R.; Kandel, Eugene S.; Hay, Nissim; Byzova, Tatiana V.

    2009-01-01

    Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1−/− fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin β1 blocking antibodies, treatment with β1-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1−/− fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin α5β1. PMID:17562714

  19. AKT mediated glycolytic shift regulates autophagy in classically activated macrophages.

    PubMed

    Matta, Sumit Kumar; Kumar, Dhiraj

    2015-09-01

    Autophagy is considered as an innate defense mechanism primarily due to its role in the targeting of intracellular pathogens for lysosomal degradation. Here we report inhibition of autophagy as an adaptive response in classically activated macrophages that helps achieve high cellular ROS production and cell death-another hallmark of innate mechanisms. We show prolonged classical activation of Raw 264.7 macrophages by treating them with IFN-γ and LPS inhibited autophagy. The inhibition of autophagy was dependent on nitric oxide (NO) production which activated the AKT-mTOR signaling, the known negative regulators of autophagy. Autophagy inhibition in these cells was accompanied with a shift to aerobic glycolysis along with a decline in the mitochondrial membrane potential (MOMP). The decline in MOMP coupled with autophagy inhibition led to increased mitochondrial content and considerably elevated cellular ROS, eventually causing cell death. Next, using specific siRNA mediated knockdowns we show AKT was responsible for the glycolytic shift and autophagy inhibition in activated macrophages. Surprisingly, AKT knockdown in activated macrophages also rescued them from cell death. Finally we show that AKT mediated autophagy inhibition in the activated macrophages correlated with the depletion of glucose from the extracellular medium, and glucose supplementation not only rescued autophagy levels and reversed other phenotypes of activated macrophages, but also inhibited cell death. Thus we report here a novel link between AKT mediated glycolytic metabolism and autophagy in the activated macrophages, and provide a possible mechanism for sustained macrophage activation in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Impaired translocation and activation of mitochondrial Akt1 mitigated mitochondrial oxidative phosphorylation Complex V activity in diabetic myocardium.

    PubMed

    Yang, Jia-Ying; Deng, Wu; Chen, Yumay; Fan, Weiwei; Baldwin, Kenneth M; Jope, Richard S; Wallace, Douglas C; Wang, Ping H

    2013-06-01

    Insulin can translocate Akt to mitochondria in cardiac muscle. The goals of this study were to define sub-mitochondrial localization of the translocated Akt, to dissect the effects of insulin on Akt isoform translocation, and to determine the direct effect of mitochondrial Akt activation on Complex V activity in normal and diabetic myocardium. The translocated Akt sequentially localized to the mitochondrial intermembrane space, inner membrane, and matrix. To confirm Akt translocation, in vitro import assay showed rapid entry of Akt into mitochondria. Akt isoforms were differentially regulated by insulin stimulation, only Akt1 translocated into mitochondria. In the insulin-resistant Type 2 diabetes model, Akt1 translocation was blunted. Mitochondrial activation of Akt1 increased Complex V activity by 24% in normal myocardium in vivo and restored Complex V activity in diabetic myocardium. Basal mitochondrial Complex V activity was lower by 22% in the Akt1(-/-) myocardium. Insulin-stimulated Complex V activity was not impaired in the Akt1(-/-) myocardium, due to compensatory translocation of Akt2 to mitochondria. Akt1 is the primary isoform that relayed insulin signaling to mitochondria and modulated mitochondrial Complex V activity. Activation of mitochondrial Akt1 enhanced ATP production and increased phosphocreatine in cardiac muscle cells. Dysregulation of this signal pathway might impair mitochondrial bioenergetics in diabetic myocardium.

  1. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  2. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  3. Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK.

    PubMed

    Dupont, Erwan; Cieniewski-Bernard, Caroline; Bastide, Bruno; Stevens, Laurence

    2011-02-01

    Our aim was to analyze the role of phosphatidylinositol 3-kinase (PI3K)-AKT and MAPK signaling pathways in the regulation of muscle mass and slow-to-fast phenotype transition during hindlimb unloading (HU). For that purpose, we studied, in rat slow soleus and fast extensor digitorum longus muscles, the time course of anabolic PI3K-AKT-mammalian target of rapamycin, catabolic PI3K-AKT-forkhead box O (FOXO), and MAPK signaling pathway activation after 7, 14, and 28 days of HU. Moreover, we performed chronic low-frequency soleus electrostimulation during HU to maintain exclusively contractile phenotype and so to determine more precisely the role of these signaling pathways in the modulation of muscle mass. HU induced a downregulation of the anabolic AKT, mammalian target of rapamycin, 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, and glycogen synthase kinase-3β targets, and an upregulation of the catabolic FOXO1 and muscle-specific RING finger protein-1 targets correlated with soleus muscle atrophy. Unexpectedly, soleus electrostimulation maintained 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, FOXO1, and muscle-specific RING finger protein-1 to control levels, but failed to reduce muscle atrophy. HU decreased ERK phosphorylation, while electrostimulation enabled the maintenance of ERK phosphorylation similar to control level. Moreover, slow-to-fast myosin heavy chain phenotype transition and upregulated glycolytic metabolism were prevented by soleus electrostimulation during HU. Taken together, our data demonstrated that the processes responsible for gradual disuse muscle plasticity in HU conditions involved both PI3-AKT and MAPK pathways. Moreover, electrostimulation during HU restored PI3K-AKT activation without counteracting soleus atrophy, suggesting the involvement of other signaling pathways. Finally, electrostimulation maintained initial contractile and metabolism properties in parallel to ERK activation, reinforcing the idea of a

  4. Activation of Akt as a mechanism for tumor immune evasion.

    PubMed

    Noh, Kyung Hee; Kang, Tae Heung; Kim, Jin Hee; Pai, Sara I; Lin, Ken Y; Hung, Chien-Fu; Wu, T-C; Kim, Tae Woo

    2009-03-01

    Immune evasion is an important reason why the immune system cannot control tumor growth. To elucidate the mechanism for tumor immune evasion, we generated an immune-resistant human papillomavirus type 16 (HPV-16) E7-expressing tumor cell line by subjecting a susceptible tumor cell line to multiple rounds of in vivo immune selection with an E7-specific vaccine. Comparison of parental and immune-resistant tumors revealed that Akt is highly activated in the immune-resistant tumors. Retroviral transfer of a constitutively active form of Akt into the parental tumor significantly increased its resistance against E7-specific CD8(+) T-cell mediated apoptosis. The observed resistance against apoptosis was found to be associated with the upregulation of antiapoptotic molecules. We also observed that intratumoral injection of an Akt inhibitor enhanced the therapeutic efficacy of E7-specific vaccine or E7-specific CD8(+) T-cell adoptive transfer against the immune-resistant tumors. Thus, our data indicate that the activation of PI3K/Akt pathway represents a new mechanism of immune escape and has important implications for the development of a novel strategy in cancer immunotherapy against immune-resistant tumor cells.

  5. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells.

    PubMed

    West, Kip A; Brognard, John; Clark, Amy S; Linnoila, Ilona R; Yang, Xiaowei; Swain, Sandra M; Harris, Curtis; Belinsky, Steven; Dennis, Phillip A

    2003-01-01

    Tobacco-related diseases such as lung cancer cause over 4.2 million deaths annually, with approximately 400,000 deaths per year occurring in the US. Genotoxic effects of tobacco components have been described, but effects on signaling pathways in normal cells have not been described. Here, we show activation of the serine/threonine kinase Akt in nonimmortalized human airway epithelial cells in vitro by two components of cigarette smoke, nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Activation of Akt by nicotine or NNK occurred within minutes at concentrations achievable by smokers and depended upon alpha(3)-/alpha(4)-containing or alpha(7)-containing nicotinic acetylcholine receptors, respectively. Activated Akt increased phosphorylation of downstream substrates such as GSK-3, p70(S6K), 4EBP-1, and FKHR. Treatment with nicotine or NNK attenuated apoptosis caused by etoposide, ultraviolet irradiation, or hydrogen peroxide and partially induced a transformed phenotype manifest as loss of contact inhibition and loss of dependence on exogenous growth factors or adherence to ECM. In vivo, active Akt was detected in airway epithelial cells and lung tumors from NNK-treated A/J mice, and in human lung cancers derived from smokers. Redundant Akt activation by nicotine and NNK could contribute to tobacco-related carcinogenesis by regulating two processes critical for tumorigenesis, cell growth and apoptosis.

  6. Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: a rat model for mania.

    PubMed

    Yu, Hyun-Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2010-08-16

    Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na-K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1mM of ouabain. Phosphorylation of GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8h after a single 1mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  7. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes.

    PubMed

    Choudhary, Chunaram; Olsen, Jesper V; Brandts, Christian; Cox, Jürgen; Reddy, Pavankumar N G; Böhmer, Frank D; Gerke, Volker; Schmidt-Arras, Dirk-E; Berdel, Wolfgang E; Müller-Tidow, Carsten; Mann, Matthias; Serve, Hubert

    2009-10-23

    Inappropriate activation of oncogenic kinases at intracellular locations is frequently observed in human cancers, but its effects on global signaling are incompletely understood. Here, we show that the oncogenic mutant of Flt3 (Flt3-ITD), when localized at the endoplasmic reticulum (ER), aberrantly activates STAT5 and upregulates its targets, Pim-1/2, but fails to activate PI3K and MAPK signaling. Conversely, membrane targeting of Flt3-ITD strongly activates the MAPK and PI3K pathways, with diminished phosphorylation of STAT5. Global phosphoproteomics quantified 12,186 phosphorylation sites, confirmed compartment-dependent activation of these pathways and discovered many additional components of Flt3-ITD signaling. The differential activation of Akt and Pim kinases by ER-retained Flt3-ITD helped to identify their putative targets. Surprisingly, we find spatial regulation of tyrosine phosphorylation patterns of the receptor itself. Thus, intracellular activation of RTKs by oncogenic mutations in the biosynthetic route may exploit cellular architecture to initiate aberrant signaling cascades, thus evading negative regulation.

  8. Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

    PubMed Central

    Addison, Christina L.; Dimitroulakos, Jim

    2010-01-01

    Background In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. Methodology/Principal Findings The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. Conclusions/Significance These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors. PMID:20838437

  9. Endomembrane PtdIns(3,4,5)P3 activates the PI3K-Akt pathway.

    PubMed

    Jethwa, Nirmal; Chung, Gary H C; Lete, Marta G; Alonso, Alicia; Byrne, Richard D; Calleja, Véronique; Larijani, Banafshé

    2015-09-15

    PKB/Akt activation is a common step in tumour growth, proliferation and survival. Akt activation is understood to occur at the plasma membrane of cells in response to growth factor stimulation and local production of the phosphoinositide lipid phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] following phosphoinositide 3-kinase (PI3K) activation. The metabolism and turnover of phosphoinositides is complex--they act as signalling molecules as well as structural components of biological membranes. The localisation and significance of internal pools of PtdIns(3,4,5)P3 has long been speculated upon. By using transfected and recombinant protein probes for PtdIns(3,4,5)P3, we show that PtdIns(3,4,5)P3 is enriched in the nuclear envelope and early endosomes. By exploiting an inducible dimerisation device to recruit Akt to these compartments, we demonstrate that Akt can be locally activated in a PtdIns(3,4,5)P3-dependent manner and has the potential to phosphorylate compartmentally localised downstream substrates. This could be an important mechanism to regulate Akt isoform substrate specificity or influence the timing and duration of PI3K pathway signalling. Defects in phosphoinositide metabolism and localisation are known to contribute to cancer, suggesting that interactions at subcellular compartments might be worthwhile targets for therapeutic intervention.

  10. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells

    PubMed Central

    Li, Hui-Fang; Kim, Jung-Sik; Waldman, Todd

    2009-01-01

    Background Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance. Methods Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays. Results Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells. Conclusion These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients. PMID:19828040

  11. Constitutive Activation of AKT2 in Humans Leads to Hypoglycemia Without Fatty Liver or Metabolic Dyslipidemia.

    PubMed

    Minic, Marina; Rocha, Nuno; Harris, Julie; Groeneveld, Matthijs P; Leiter, Sarah; Wareham, Nicholas; Sleigh, Alison; De Lonlay, Pascale; Hussain, Khalid; O'Rahilly, Stephen; Semple, Robert K

    2017-08-01

    The activating p.Glu17Lys mutation in AKT2, a kinase mediating many of insulin's metabolic actions, causes hypoinsulinemic hypoglycemia and left-sided hemihypertrophy. The wider metabolic profile and longer-term natural history of the condition has not yet been reported. To characterize the metabolic and cellular consequences of the AKT2 p.Glu17Lys mutation in two previously reported males at the age of 17 years. Body composition analysis using dual-energy X-ray absorptiometry, overnight profiling of plasma glucose, insulin, and fatty acids, oral glucose tolerance testing, and magnetic resonance spectroscopy to determine hepatic triglyceride content was undertaken. Hepatic de novo lipogenesis was quantified using deuterium incorporation into palmitate. Signaling in dermal fibroblasts was studied ex vivo. Both patients had 37% adiposity. One developed hypoglycemia after 2 hours of overnight fasting with concomitant suppression of plasma fatty acids and ketones, whereas the other maintained euglycemia with an increase in free fatty acids. Blood glucose excursions after oral glucose were normal in both patients, albeit with low plasma insulin concentrations. In both patients, plasma triglyceride concentration, hepatic triglyceride content, and fasting hepatic de novo lipogenesis were normal. Dermal fibroblasts of one proband showed low-level constitutive phosphorylation of AKT and some downstream substrates, but no increased cell proliferation rate. The p.Glu17Lys mutation of AKT2 confers low-level constitutive activity upon the kinase and produces hypoglycemia with suppressed fatty acid release from adipose tissue, but not fatty liver, hypertriglyceridemia, or elevated hepatic de novo lipogenesis. Hypoglycemia may spontaneously remit.

  12. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2008-03-01

    ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Martin H. Hager 5e. TASK NUMBER Email: Martin.Hager@childrens.harvard.edu 5f. WORK UNIT...SUPPLEMENTARY NOTES 14. ABSTRACT This project focuses on the novel finding from our group that the serine-threonine kinase Akt1 partitions into...signaling protein that is frequently activated in prostate cancer. I have hypothesized in this project that cholesterol accumulation in prostate

  13. ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages.

    PubMed

    Gozal, E; Forman, H J; Torres, M

    2001-09-01

    Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP > ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.

  14. Pharmacological Inhibition of PERK Attenuates Early Brain Injury After Subarachnoid Hemorrhage in Rats Through the Activation of Akt.

    PubMed

    Yan, Feng; Cao, Shenglong; Li, Jianru; Dixon, Brandon; Yu, Xiaobo; Chen, Jingyin; Gu, Chi; Lin, Wang; Chen, Gao

    2017-04-01

    Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Endoplasmic reticulum (ER) stress was reported to have a vital role in the pathophysiology of neuronal apoptosis in the brain. The present study was designed to investigate the potential effects of ER stress and its downstream signals in early brain injury after SAH. One hundred thirty-four rats were subjected to an endovascular perforation model of SAH. The RNA-activated protein kinase-like ER kinase (PERK) inhibitor GSK2606414 and the Akt inhibitor MK2206 were injected intracerebroventricularly. SAH grade, neurologic scores, and brain water content were measured 72 h after subarachnoid hemorrhage. Expression of PERK and its downstream signals, Akt, Bcl-2, Bax, and cleaved caspase-3, were examined using Western blot analysis. Specific cell types that expressed PERK were detected with double immunofluorescence staining. Neuronal cell death was demonstrated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Our results showed that the expression of p-PERK and its downstream targets, p-eIF2α and ATF4, increased after SAH and peaked at 72 h after SAH. PERK was expressed mostly in neurons. The inhibition of PERK with GSK2606414 reduced p-PERK, p-eIF2α, and ATF4 expression. Furthermore, GSK2606414 treatment increased p-Akt levels and the Bcl-2/Bax ratio as well as decreased cleaved caspase-3 expression and neuronal death, thereby improving neurological deficits at 72 h after SAH. The selective Akt inhibitor MK2206 abolished the beneficial effects of GSK2606414. PERK, the major transducer of ER stress, is involved in neuronal apoptosis after SAH. The inhibition of PERK reduces early brain injury via Akt-related anti-apoptosis pathways. PERK may serve as a promising target for future therapeutic intervention.

  15. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  16. The critical role of Akt in cardiovascular function.

    PubMed

    Abeyrathna, Prasanna; Su, Yunchao

    2015-11-01

    Akt kinase, a member of AGC kinases, is important in many cellular functions including proliferation, migration, cell growth and metabolism. There are three known Akt isoforms which play critical and diverse roles in the cardiovascular system. Akt activity is regulated by its upstream regulatory pathways at transcriptional and post-translational levels. Beta-catenin/Tcf-4, GLI1 and Stat-3 are some of few known transcriptional regulators of AKT gene. Threonine 308 and serine 473 are the two critical phosphorylation sites of Akt1. Translocation of Akt to the cell membrane facilitates PDK1 phosphorylation of the threonine site. The serine site is phosphorylated by mTORC2. Ack1, Src, PTK6, TBK1, IKBKE and IKKε are some of the non-canonical pathways which affect the Akt activity. Protein-protein interactions of Akt to actin and Hsp90 increase the Akt activity while Akt binding to other proteins such as CTMP and TRB3 reduces the Akt activity. The action of Akt on its downstream targets determines its function in cardiovascular processes such as cell survival, growth, proliferation, angiogenesis, vasorelaxation, and cell metabolism. Akt promotes cell survival via caspase-9, YAP, Bcl-2, and Bcl-x activities. Inhibition of FoxO proteins by Akt also increases cell survival by transcriptional mechanisms. Akt stimulates cell growth and proliferation through mTORC1. Akt also increases VEGF secretion and mediates eNOS phosphorylation, vasorelaxation and angiogenesis. Akt can increase cellular metabolism through its downstream targets GSK3 and GLUT4. The alterations of Akt signaling play an important role in many cardiovascular pathological processes such as atherosclerosis, cardiac hypertrophy, and vascular remodeling. Several Akt inhibitors have been developed and tested as anti-tumor agents. They could be potential novel therapeutics for the cardiovascular diseases.

  17. AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1.

    PubMed

    Fischer, Adrian; Weber, Wilfried; Warscheid, Bettina; Radziwill, Gerald

    2017-01-01

    Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Modified acoustic transmission tube apparatus incorporating an active downstream termination.

    PubMed

    Machuca-Tzili, F Arturo; Orduña-Bustamante, Felipe; Pérez-López, Antonio; Pérez-Ruiz, Santiago J; Pérez-Matzumoto, Andrés E

    2017-02-01

    Current techniques for measuring normal incidence sound transmission loss with a modified impedance tube, or transmission tube, require setting up two different absorbing termination loads at the end of the downstream tube [ASTM E2611-09, Standard Test Method for Measurement of Normal Incidence Sound Transmission of Acoustical Materials Based on the Transfer Matrix Method (American Society for Testing and Materials, West Conshohocken, 2009)]. The process of physically handling the two required passive absorbing loads is a possible source of measurement errors, which are mainly due to changes in sample test position, or in test setup re-assembly, between measurements. In this paper, a modified transmission tube apparatus is proposed for non-intrusively changing the downstream acoustic load by means of a combined passive-active termination. It provides a controlled variable sound absorption which simplifies the setup of standard two-load techniques, without the need of physically handling the apparatus during the tests. This virtually eliminates the risk of errors associated with the physical manipulation of the two passive terminations. Transmission loss measurements in some representative test conditions are reported, showing improvements over current implementations, in reducing by approximately 50% the measurement variations associated with the setup of the two required absorbing terminations. Measurement results agree within 0.4 dB (maximum difference in high resolution broadband), and 0.04 dB (mean difference in 1/3-octave bands), with those obtained using standard passive two-load methods.

  19. DMH1 increases glucose metabolism through activating Akt in L6 rat skeletal muscle cells.

    PubMed

    Xie, Xin; Xu, Xiao-Ming; Li, Na; Zhang, Yong-Hui; Zhao, Yu; Ma, Chun-Yan; Dong, De-Li

    2014-01-01

    DMH1(4-[6-(4-Isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a compound C analogue with the structural modifications at the 3- and 6-positions in pyrazolo[1,5-a]pyrimidine backbone. Compound C was reported to inhibit both AMPK and Akt. Our preliminary work found that DMH1 activated Akt. Since Akt was involved in glucose metabolism, we aimed to identify the effects of DMH1 on glucose metabolism in L6 rat muscle cells and the potential mechanism. Results showed that DMH1 increased lactic acid release and glucose consumption in L6 rat muscle cells in a dose-dependent manner. DMH1 activated Akt in L6 cells. Akt inhibitor inhibited DMH1-induced Akt activation and DMH1-induced increases of glucose uptake and consumption. DMH1 had no cytotoxicity in L6 cells, but inhibited mitochondrial function and reduced ATP production. DMH1 showed no effect on AMPK, but in the presence of Akt inhibitor, DMH1 significantly activated AMPK. Compound C inhibited DMH1-induced Akt activation in L6 cells. Compound C inhibited DMH1-induced increase of glucose uptake, consumption and lactic acid release in L6 cells. DMH1 inhibited PP2A activity, and PP2A activator forskolin reversed DMH1-induced Akt activation. We concluded that DMH1 increased glucose metabolism through activating Akt and DMH1 activated Akt through inhibiting PP2A activity in L6 rat muscle cells. In view of the analogue structure of DMH1 and compound C and the contrasting effects of DMH1 and compound C on Akt, the present study provides a novel leading chemical structure targeting Akt with potential use for regulating glucose metabolism.

  20. BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

    PubMed Central

    Urban, Bettina C; Collard, Tracey J; Eagle, Catherine J; Southern, Samantha L; Greenhough, Alexander; Hamdollah-Zadeh, Maryam; Ghosh, Anil; Paraskeva, Christos; Silver, Andrew; Williams, Ann C

    2016-01-01

    Objective Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. Design Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. Results We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3β and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. Conclusions Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy. PMID:26033966

  1. Distinct roles of AKT isoforms in regulating β1-integrin activity, migration, and invasion in prostate cancer

    PubMed Central

    Virtakoivu, Reetta; Pellinen, Teijo; Rantala, Juha K.; Perälä, Merja; Ivaska, Johanna

    2012-01-01

    AKT1 and AKT2 kinases have been shown to play opposite roles in breast cancer migration and invasion. In this study, an RNA interference screen for integrin activity inhibitors identified AKT1 as an inhibitor of β1-integrin activity in prostate cancer. Validation experiments investigating all three AKT isoforms demonstrated that, unlike in breast cancer, both AKT1 and AKT2 function as negative regulators of cell migration and invasion in PC3 prostate cancer cells. Down-regulation of AKT1 and AKT2, but not AKT3, induced activation of cell surface β1-integrins and enhanced adhesion, migration, and invasion. Silencing of AKT1 and AKT2 also resulted in increased focal adhesion size. Importantly, the mechanisms involved in integrin activity regulation were distinct for the two AKT isoforms. Silencing of AKT1 relieved feedback suppression of the expression and activity of several receptor tyrosine kinases, including EGFR and MET, with established cross-talk with β1-integrins. Silencing of AKT2, on the other hand, induced up-regulation of the microRNA-200 (miR-200) family, and overexpression of miR-200 was sufficient to induce integrin activity and cell migration in PC3 cells. Taken together, these data define an inhibitory role for both AKT1 and AKT2 in prostate cancer migration and invasion and highlight the cell type–specific actions of AKT kinases in the regulation of cell motility. PMID:22809628

  2. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    PubMed Central

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

  3. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity.

    PubMed

    Oeck, S; Al-Refae, K; Riffkin, H; Wiel, G; Handrick, R; Klein, D; Iliakis, G; Jendrossek, V

    2017-02-17

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition.

  4. Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2007-03-01

    Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

  5. Akt phosphorylates myc-associated zinc finger protein (MAZ), releases P-MAZ from the p53 promoter, and activates p53 transcription.

    PubMed

    Lee, Wei-Ping; Lan, Keng-Hsin; Li, Chung-Pin; Chao, Yee; Lin, Han-Chieh; Lee, Shou-Dong

    2016-05-28

    The p53 protein is a cell cycle regulator. When the cell cycle progresses, p53 plays an important role in putting a brake on the G1 phase to prevent unwanted errors during cell division. Akt is a downstream kinase of receptor tyrosine kinase. Upon activation, Akt phorphorylates IKK that then phosphorylates IκB and releases NF-κB, leading to transcriptional activation of Dmp1. Dmp1 is a transcriptional activator of Arf. It has been known that oncogene activation stabilizes p53 through transcriptional activation of Arf, which then binds and inhibits Mdm2. In the current study, we show that myc-associated zinc finger protein (MAZ) is a transcriptional repressor of the p53 promoter. Akt phosphorylates MAZ at Thr385, and the phosphorylated MAZ is released from the p53 promoter, leading to transcriptional activation of p53, a new mechanism that contributes to increased p53 protein pool during oncogene activation.

  6. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  7. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  8. Insulin-mediated signaling promotes proliferation and survival of glioblastoma through Akt activation

    PubMed Central

    Gong, Yuanying; Ma, Yufang; Sinyuk, Maksim; Loganathan, Sudan; Thompson, Reid C.; Sarkaria, Jann N.; Chen, Wenbiao; Lathia, Justin D.; Mobley, Bret C.; Clark, Stephen W.; Wang, Jialiang

    2016-01-01

    Background Metabolic complications such as obesity, hyperglycemia, and type 2 diabetes are associated with poor outcomes in patients with glioblastoma. To control peritumoral edema, use of chronic high-dose steroids in glioblastoma patients is common, which can result in de novo diabetic symptoms. These metabolic complications may affect tumors via profound mechanisms, including activation of insulin receptor (InsR) and the related insulin-like growth factor 1 receptor (IGF1R) in malignant cells. Methods In the present study, we assessed expression of InsR in glioblastoma surgical specimens and glioblastoma response to insulin at physiologically relevant concentrations. We further determined whether genetic or pharmacological targeting of InsR affected oncogenic functions of glioblastoma in vitro and in vivo. Results We showed that InsR was commonly expressed in glioblastoma surgical specimens and xenograft tumor lines, with mitogenic isoform-A predominating. Insulin at physiologically relevant concentrations promoted glioblastoma cell growth and survival, potentially via Akt activation. Depletion of InsR impaired cellular functions and repressed orthotopic tumor growth. The absence of InsR compromised downstream Akt activity, but yet stimulated IGF1R expression. Targeting both InsR and IGF1R with dual kinase inhibitors resulted in effective blockade of downstream signaling, loss of cell viability, and repression of xenograft tumor growth. Conclusions Taken together, our work suggests that glioblastoma is sensitive to the mitogenic functions of insulin, thus significant insulin exposure imposes risks to glioblastoma patients. Additionally, dual inhibition of InsR and IGF1R exhibits promise for treating glioblastoma. PMID:26136493

  9. Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

    PubMed

    Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

    2014-01-01

    Membrane type 1-matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.

  10. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  11. SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    PubMed Central

    Yoshioka, Yuichiro; Suzuki, Takehiro; Matsuo, Yo; Nakakido, Makoto; Tsurita, Giichiro; Simone, Cristiano; Watanabe, Toshiaki; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy. PMID:27626683

  12. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells.

    PubMed

    Ghosh Choudhury, Goutam; Lenin, Mahimainathan; Calhaun, Cheresa; Zhang, Jian-Hua; Abboud, Hanna E

    2003-02-01

    Regulation of the forkhead domain transcription factors by PDGF has not been studied. In this report, we investigated the role of PDGF-induced Akt in regulating forkhead domain protein FKHRL1 in glomerular mesangial cells. PDGF increased phosphorylation of FKHRL1 in a time- and PI 3 kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced FKHRL1 phosphorylation. PDGF inhibited transcription of a forkhead DNA binding element-driven reporter gene. This inhibition was mimicked by constitutively active myristoylated Akt. Moreover, FKHR1-mediated transcription of the reporter gene was completely attenuated by both PDGF and Myr-Akt. One of the targets of forkhead transcription factors is the proapoptotic Fas ligand (FasL) gene. PDGF, as well as Myr-Akt, inhibited transcription of FasL. In contrast, inhibition of PI 3 kinase and dominant negative Akt increased FasL gene transcription, suggesting that suppression of PI 3 kinase/Akt signalling may induce apoptosis in mesangial cells via upregulation of FasL expression. However, expression of dominant negative Akt by adenovirus did not induce apoptosis in mesangial cells, suggesting that Akt-independent antiapoptotic mechanisms also exist. Together, our data demonstrate for the first time that PDGF inactivates forkhead domain transcription factor by Akt-dependent phosphorylation and that suppression of Akt signalling is not sufficient to induce apoptosis in mesangial cells.

  13. Contactin-1 reduces E-cadherin expression via activating AKT in lung cancer.

    PubMed

    Yan, Judy; Wong, Nicholas; Hung, Claudia; Chen, Wendy Xin-Yi; Tang, Damu

    2013-01-01

    Contactin-1 has been shown to promote cancer metastasis. However, the underlying mechanisms remain unclear. We report here that knockdown of contactin-1 in A549 lung cancer cells reduced A549 cell invasion and the cell's ability to grow in soft agar without affecting cell proliferation. Reduction of contactin-1 resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. In an effort to investigate the mechanisms whereby contactin-1 reduces E-cadherin expression, we observed that contactin-1 plays a role in AKT activation, as knockdown of contactin-1 attenuated AKT activation. Additionally, inhibition of AKT activation significantly enhanced E-cadherin expression, an observation that mimics the situation observed in contactin-1 knockdown, suggesting that activation of AKT plays a role in contactin-1-mediated downregulation of E-cadherin. In addition, we were able to show that knockdown of contactin-1 did not further reduce A549 cell's invasion ability, when AKT activation was inhibited by an AKT inhibitor. To further support our findings, we overexpressed CNTN-1 in two CNTN-1 null breast cancer cell lines expressing E-cadherin. Upon overexpression, CNTN-1 reduced E-cadherin levels in one cell line and increased AKT activation in the other. Furthermore, in our study of 63 primary lung cancers, we observed 65% of primary lung cancers being contactin-1 positive and in these carcinomas, 61% were E-cadherin negative. Collectively, we provide evidence that contactin-1 plays a role in the downregulation of E-cadherin in lung cancer and that AKT activation contributes to this process. In a study of mechanisms responsible for contactin-1 to activate AKT, we demonstrated that knockdown of CNTN-1 in A549 cells did not enhance PTEN expression but upregulated PHLPP2, a phosphatase that dephosphorylates AKT. These observations thus suggest that contactin-1 enhances AKT activation in part by preventing PHLPP2-mediated AKT

  14. Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung.

    PubMed

    Malanga, Donatella; Scrima, Marianna; De Marco, Carmela; Fabiani, Fernanda; De Rosa, Nicla; De Gisi, Silvia; Malara, Natalia; Savino, Rocco; Rocco, Gaetano; Chiappetta, Gennaro; Franco, Renato; Tirino, Virginia; Pirozzi, Giuseppe; Viglietto, Giuseppe

    2008-03-01

    Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G-->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

  15. Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.

    PubMed

    Zuo, Tao; Liu, Ta-Ming; Lan, Xun; Weng, Yu-I; Shen, Rulong; Gu, Fei; Huang, Yi-Wen; Liyanarachchi, Sandya; Deatherage, Daniel E; Hsu, Pei-Yin; Taslim, Cenny; Ramaswamy, Bhuvaneswari; Shapiro, Charles L; Lin, Huey-Jen L; Cheng, Alfred S L; Jin, Victor X; Huang, Tim H-M

    2011-03-01

    Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.

  16. Epigenetic Silencing Mediated Through Activated PI3K/AKT Signaling in Breast Cancer

    PubMed Central

    Zuo, Tao; Liu, Ta-Ming; Lan, Xun; Weng, Yu-I; Shen, Rulong; Gu, Fei; Huang, Yi-Wen; Liyanarachchi, Sandya; Deatherage, Daniel E.; Hsu, Pei-Yin; Taslim, Cenny; Ramaswamy, Bhuvaneswari; Shapiro, Charles L.; Lin, Huey-Jen L.; Cheng, Alfred S.L.; Jin, Victor X.; Huang, Tim H.-M.

    2011-01-01

    Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future. PMID:21216892

  17. Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    PubMed Central

    Yoon, Heejeong; Kim, Deog Joong; Ahn, Eun Hyun; Gellert, Ginelle C.; Shay, Jerry W.; Ahn, Chang-Ho; Lee, Young Bok

    2010-01-01

    The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various types of human cancer cells. Combined treatment of Akt1 AO with several cytotoxic drugs resulted in an additive growth inhibition of Caki-1 cells. The in vivo effectiveness of Akt1 AO was determined using two different xenograft nude mouse models. Akt1 AO (30 mg/kg, i.v. every 48 h) significantly inhibited the tumor growth of nude mouse subcutaneously implanted with U251 human glioblastoma cells after 27 days treatment. Akt1 AO (30 mg/kg, i.p continuously via osmotic pump) also significantly inhibited the tumor formation in nude mice implanted with luciferase-expressing MIA human pancreatic cancer cells (MIA-Luc) after 14 days of treatment. The luciferase signals from MIA-Luc cells were reduced or completely abolished after 2 weeks of treatment and the implanted tumors were barely detectable. Our findings suggest that Akt1 AO alone or in combination with other clinically approved anticancer agents should be further explored and progressed into clinical studies as a potential novel therapeutic agent. PMID:19693774

  18. RAS and downstream RAF-MEK and PI3K-AKT signaling in neuronal development, function and dysfunction

    PubMed Central

    Zhong, Jian

    2016-01-01

    In postmitotic neurons, the activation of RAS family small GTPases regulates survival, growth and differentiation. Dysregulation of RAS or its major effector pathway, the cascade of RAF-, mitogen-activated and extracellular-signal regulated kinase kinases (MEK), and extracellular-signal regulated kinases (ERK) causes the Rasopathies, a group of neurodevelopmental disorders whose pathogenic mechanisms are the subject of intense research. I here summarize the functions of RAS – RAF – MEK – ERK signaling in neurons in vivo, and discuss perspectives for harnessing this pathway to enable novel treatments for nervous system injury, the Rasopathies, and possibly other neurological conditions. PMID:26760308

  19. The neurotensin gene is a downstream target for Ras activation.

    PubMed Central

    Evers, B M; Zhou, Z; Celano, P; Li, J

    1995-01-01

    Ras regulates novel patterns of gene expression and the differentiation of various eukaryotic cell types. Stable transfection of Ha-ras into the human colon cancer line CaCo2 results in the morphologic differentiation to a small bowel phenotype. The purpose of our study was to determine whether the Ras regulatory pathway plays a role in the expression of the neurotensin gene (NT/N), a terminally differentiated endocrine product specifically localized in the gastrointestinal tract to the adult small bowel. We found that CaCo2-ras cells, but not parental CaCo2, express high levels of the human NT/N gene and, moreover, that this increase in gene expression is regulated at the level of transcription. Transfection experiments using NT/N-CAT mutation constructs identify the proximal 200 bp of NT/N flanking sequence as sufficient for maximal Ras-mediated NT/N reporter gene induction. Furthermore, a proximal AP-1/CRE motif is crucial for this Ras-mediated NT/N activation. Wild-type Ha-ras induces NT/N gene expression, albeit at lower levels than activated Ras; a dominant-negative Raf blocks this NT/N induction, suggesting that Raf lies down-stream of Ras in this pathway. In addition, postconfluent cultures of CaCo2 cells, which are differentiated to a small bowel phenotype, express the NT/N gene by 6 d after reaching confluency; this increase of NT/N expression is associated with concomitant increases of cellular p21ras protein. We conclude that Ras (both wild-type and activated) enhances expression of the NT/N gene in the gut-derived CaCo2 cell line, suggesting an important role for the Ras signaling pathway in NT/N gene transcription. Our results underscore the possibility that tissue-specific genes (such as NT/N) expressed in distinct subpopulations of the gut may be subject to Ras regulation. Finally, we speculate that the NT/N gene and the CaCo2 and CaCo2-ras cell systems will provide unique models to further define the cellular mechanisms leading to mammalian

  20. Gastrointestinal growth factors and hormones have divergent effects on Akt activation

    PubMed Central

    Berna, Marc J.; Tapia, Jose A.; Sancho, Veronica; Thill, Michelle; Pace, Andrea; Hoffmann, K. Martin; Gonzalez-Fernandez, Lauro; Jensen, Robert T.

    2009-01-01

    Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigate in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters[CCK, bombesin,carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations(pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations(nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory

  1. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    SciTech Connect

    Ohashi, Kazuya; Nagata, Yosuke; Wada, Eiji; Zammit, Peter S.; Shiozuka, Masataka; Matsuda, Ryoichi

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  2. Beer elicits vasculoprotective effects through Akt/eNOS activation.

    PubMed

    Vilahur, Gemma; Casani, Laura; Mendieta, Guiomar; Lamuela-Raventos, Rosa M; Estruch, Ramon; Badimon, Lina

    2014-12-01

    There is controversy regarding the effect of alcohol beverage intake in vascular vasodilatory function in peripheral arteries. The effects of beer intake in coronary vasodilation remain unknown. We investigated whether regular beer intake (alcohol and alcohol-free) protects against hypercholesterolaemia-induced coronary endothelial dysfunction and the mechanisms behind this effect. Pigs were fed 10 days: (i) a Western-type hypercholesterolaemic diet (WD); (ii) WD+low-dose beer (12·5 g alcohol/day); (iii) WD+moderate-dose beer (25 g alcohol/day); or (iv) WD+moderate-dose alcohol-free-beer (0·0 g alcohol/day). Coronary responses to endothelium-dependent vasoactive drugs (acetylcholine: receptor mediated; calcium ionophore-A23189: nonreceptor mediated), endothelium-independent vasoactive drug (SNP) and L-NMMA (NOS-antagonist) were evaluated in the LAD coronary artery by flow Doppler. Coronary Akt/eNOS activation, MCP-1 expression, oxidative DNA damage and superoxide production were assessed. Lipid profile, lipoproteins resistance to oxidation and urinary isoxanthohumol concentration were evaluated. Alcoholic and nonalcoholic beer intake prevented WD-induced impairment of receptor- and non-receptor-operated endothelial-dependent coronary vasodilation. All animals displayed a similar vasodilatory response to SNP and L-NMMA blunted all endothelial-dependent vasorelaxation responses. Haemodynamic parameters remained unchanged. Coronary arteries showed lower DNA damage and increased Akt/eNOS axis activation in beer-fed animals. Animals taking beer showed HDL with higher antioxidant capacity, higher LDL resistance to oxidation and increased isoxanthohumol levels. Weight, lipids levels, liver enzymes and MCP-1 expression were not affected by beer intake. Non-alcoholic-related beer components protect against hyperlipemia-induced coronary endothelial dysfunction by counteracting vascular oxidative damage and preserving the Akt/eNOS pathway. Light-to-moderate beer

  3. Pancreatic-β-cell survival and proliferation are promoted by protein kinase G type Iα and downstream regulation of AKT/FOXO1.

    PubMed

    Wong, Janica C; Vo, Van; Gorjala, Priyatham; Fiscus, Ronald R

    2017-09-01

    Early studies showed nitric oxide as a pro-inflammatory-cytokine-induced toxin involved in pancreatic β-cell destruction during pathogenesis of type-1 diabetes. However, nitric oxide has both cytotoxic and cytoprotective effects on mammalian cells, depending on concentration and micro-environmental surroundings. Our studies have shown that low/physiological-level nitric oxide selectively activates protein kinase G type Iα isoform, promoting cytoprotective/pro-cell-survival effects in many cell types. In bone marrow-derived stromal/mesenchymal stem cells, protein kinase G type Iα mediates autocrine effects of nitric oxide and atrial natriuretic peptide, promoting DNA-synthesis/proliferation and cell survival. In this study, endothelial nitric oxide synthase/neuronal nitric oxide synthase inhibitor L-NIO (L-N(5)-(1-iminoethyl)ornithine), soluble guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one), atrial natriuretic peptide-receptor inhibitor A71915 and protein kinase G type Iα kinase activity inhibitor DT-2 all increased apoptosis and decreased insulin secretion in RINm5F pancreatic β-cells, suggesting autocrine regulatory role for endogenous nitric oxide- and atrial natriuretic peptide-induced activation of protein kinase G type Iα. In four pancreatic β-cell lines, Beta-TC-6, RINm5F, INS-1 and 1.1B4, protein kinase G type Iα small-interfering RNA decreased phospho-serine-239-VASP (indicator of endogenous protein kinase G type Iα kinase activity), increased apoptosis and decreased proliferation. In protein kinase G type Iα-knockdown β-cell lines, expressions of phospho-protein kinase B (PKB/AKT) (AKT), phospho-Forkhead box protein O1 (FOXO1) (transcriptional repressor of pancreas duodenum homobox-1) and pancreas duodenum homobox-1 were decreased, suppressing proliferation and survival in pancreatic β-cells. The data suggest autocrine nitric oxide/atrial natriuretic peptide-induced activation of protein kinase G type Iα/p-AKT

  4. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  5. Activation of PI3K/Akt/mTOR signaling in the tumor stroma drives endocrine therapy-dependent breast tumor regression

    PubMed Central

    Polo, María Laura; Riggio, Marina; May, María; Rodríguez, María Jimena; Perrone, María Cecilia; Stallings-Mann, Melody; Kaen, Diego; Frost, Marlene; Goetz, Matthew; Boughey, Judy; Lanari, Claudia; Radisky, Derek; Novaro, Virginia

    2015-01-01

    Improved efficacy of neoadjuvant endocrine-targeting therapies in luminal breast carcinomas could be achieved with optimal use of pathway targeting agents. In a mouse model of ductal breast carcinoma we identify a tumor regressive stromal reaction that is induced by neoadjuvant endocrine therapy. This reparative reaction is characterized by tumor neovascularization accompanied by infiltration of immune cells and carcinoma-associated fibroblasts that stain for phosphorylated ribosomal protein S6 (pS6), downstream the PI3K/Akt/mTOR pathway. While tumor variants with higher PI3K/Akt/mTOR activity respond well to a combination of endocrine and PI3K/Akt/mTOR inhibitors, tumor variants with lower PI3K/Akt/mTOR activity respond more poorly to the combination therapy than to the endocrine therapy alone, associated with inhibition of stromal pS6 and the reparative reaction. In human breast cancer xenografts we confirm that such differential sensitivity to therapy is primarily determined by the level of PI3K/Akt/mTOR in tumor cells. We further show that the clinical response of breast cancer patients undergoing neoadjuvant endocrine therapy is associated with the reparative stromal reaction. We conclude that tumor level and localization of pS6 are associated with therapeutic response in breast cancer and represent biomarkers to distinguish which tumors will benefit from the incorporation of PI3K/Akt/mTOR inhibitors with neoadjuvant endocrine therapy. PMID:26098779

  6. Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

    PubMed Central

    Ridnour, Lisa A.; Barasch, Kimberly M.; Windhausen, Alisha N.; Dorsey, Tiffany H.; Lizardo, Michael M.; Yfantis, Harris G.; Lee, Dong H.; Switzer, Christopher H.; Cheng, Robert Y. S.; Heinecke, Julie L.; Brueggemann, Ernst; Hines, Harry B.; Khanna, Chand; Glynn, Sharon A.; Ambs, Stefan; Wink, David A.

    2012-01-01

    Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation. PMID:22957045

  7. Low-power laser irradiation inhibits Aβ25-35-induced cell apoptosis through Akt activation

    NASA Astrophysics Data System (ADS)

    Zhang, Zhigang; Tang, Yonghong

    2009-08-01

    Low-power laser irradiation (LPLI) can modulate various cellular processes such as proliferation, differentiation and apoptosis. Recently, LPLI has been applied to moderate Alzheimer's disease (AD), but the underlying mechanism remains unknown. The protective role of LPLI against the amyloid beta peptide (Aβ), a major constituent of AD plaques, has not been studied. PI3K/Akt pathway is extremely important in protecting cells from apoptosis caused by diverse stress stimuli. However, whether LPLI can inhibit Aβ-induced apoptosis through Akt activation is still unclear. In current study, using FRET (fluorescence resonance energy transfer) technique, we investigated the activity of Akt in response to LPLI treatment. B kinase activity reporter (BKAR), a recombinant FRET probe of Akt, was utilized to dynamically detect the activation of Akt after LPLI treatment. The results show that LPLI promoted the activation of Akt. Moreover, LPLI inhibits apoptosis induced by Aβ25-35 and the apoptosis inhibition can be abolished by wortmannin, a specific inhibitor of PI3K/Akt. Taken together, these results suggest that LPLI can inhibit Aβ25-35-induced cell apoptosis through Akt activation.

  8. Akt-dependent NF-{kappa}B activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis

    SciTech Connect

    Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S.; Wang Jianying; Raufman, Jean-Pierre

    2009-02-01

    Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-{kappa}B. Deoxycholyltaurine (DCT) treatment attenuated TNF-{alpha}-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-{kappa}B nuclear translocation and transcriptional activity (detected by NF-{kappa}B binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-{kappa}B nuclear translocation and attenuation of TNF-{alpha}-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and I{kappa}B{alpha} kinase attenuated NF-{kappa}B transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable I{kappa}B{alpha} 'super-repressor' blocked the actions of DCT on both NF-{kappa}B activation and TNF-{alpha}-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-{kappa}B transcriptional activity and TNF-{alpha}-induced apoptosis. Chemical inhibitors of Akt and NF-{kappa}B activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-{kappa}B activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation.

  9. Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1.

    PubMed

    Albury-Warren, Toya M; Pandey, Veethika; Spinel, Lina P; Masternak, Michal M; Altomare, Deborah A

    2016-01-01

    Protein kinase B/AKT has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin-responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2(-/-)) are glucose-intolerant and insulin-resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1(Myr)) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose-intolerant AKT1(Myr) mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn off the transgene caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer.

  10. The tumor suppressor protein menin inhibits AKT activation by regulating its cellular localization

    PubMed Central

    Wang, Yan; Ozawa, Atsushi; Zaman, Shadia; Prasad, Nijaguna B.; Chandrasekharappa, Settara C.; Agarwal, Sunita K.; Marx, Stephen J.

    2010-01-01

    Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of multiple endocrine organs. Mutations in the MEN1 gene that encodes for the menin protein are the predominant cause for hereditary MEN1 syndrome. Though menin is a tumor suppressor, its molecular mechanism of action has not been defined. Here we report that menin interacts with AKT1 in vitro and in vivo. Menin downregulates the level of active AKT and its kinase activity. Through interaction with AKT1, menin suppresses both AKT1 induced proliferation and anti-apoptosis in non-endocrine and endocrine cells. Confocal microscopy analysis revealed that menin regulates AKT1 in part by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation. Our findings may be generalizable to other cancers, insofar as we found that loss of menin expression was also associated with AKT activation in a mouse model of pancreatic islet adenoma. Together, our results suggest menin as an important novel negative regulator of AKT kinase activity. PMID:21127195

  11. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

    PubMed Central

    Lindhurst, Marjorie J.; Yourick, Miranda R.; Yu, Yi; Savage, Ronald E.; Ferrari, Dora; Biesecker, Leslie G.

    2015-01-01

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome. PMID:26657992

  12. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome.

    PubMed

    Lindhurst, Marjorie J; Yourick, Miranda R; Yu, Yi; Savage, Ronald E; Ferrari, Dora; Biesecker, Leslie G

    2015-12-11

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome.

  13. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  14. EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

    PubMed

    Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

    2014-05-09

    Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Erythropoietin alleviates hepatic insulin resistance via PPARγ-dependent AKT activation

    PubMed Central

    Ge, Zhijuan; Zhang, Pengzi; Hong, Ting; Tang, Sunyinyan; Meng, Ran; Bi, Yan; Zhu, Dalong

    2015-01-01

    Erythropoietin (EPO) has beneficial effects on glucose metabolism and insulin resistance. However, the mechanism underlying these effects has not yet been elucidated. This study aimed to investigate how EPO affects hepatic glucose metabolism. Here, we report that EPO administration promoted phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation in palmitic acid (PA)-treated HepG2 cells and in the liver of high-fat diet (HFD)-fed mice, whereas adenovirus-mediated silencing of the erythropoietin receptor (EPOR) blocked EPO-induced AKT signalling in HepG2 cells. Importantly, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist and PPARγ small interfering RNA (siRNA) abrogated the EPO-induced increase in p-AKT in HepG2 cells. Lentiviral vector-mediated hepatic PPARγ silencing in HFD-fed C57BL/6 mice impaired EPO-mediated increases in glucose tolerance, insulin sensitivity and hepatic AKT activation. Furthermore, EPO activated the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling pathway, and AMPKα and SIRT1 knockdown each attenuated the EPO-induced PPARγ expression and deacetylation and PPARγ-dependent AKT activation in HepG2 cells. In summary, these findings suggest that PPARγ is involved in EPO/EPOR-induced AKT activation, and targeting the PPARγ/AKT pathway via EPO may have therapeutic implications for hepatic insulin resistance and type 2 diabetes. PMID:26643367

  16. The Akt/mTOR pathway is activated in verrucous carcinoma of the oral cavity.

    PubMed

    Chaisuparat, Risa; Limpiwatana, Seehachart; Kongpanitkul, Sanida; Yodsanga, Somchai; Jham, Bruno C

    2016-09-01

    The Akt/mTOR pathway is activated in many malignancies, including oral squamous cell carcinoma (OSCC). However, the role of the Akt/mTOR pathway in oral verrucous carcinoma (OVC), a low-grade variant of OSCC, remains unknown. Thus, the objective of this study was to investigate the activation level of important markers of the Akt/mTOR pathway in OVC and to compare the results with OSCC samples. The expression of p-Akt (Thr308), p-Akt (Ser473), and p-RPS6 was evaluated by immunohistochemistry in 30 OSCC cases, 18 OVC cases, and 30 control cases (normal epithelium overlying fibromas). Statistical analysis was performed to determine the differences in protein expression between samples. All OVC cases were positive for p-Akt (Thr308), p-Akt (Ser473), and p-RPS6. There were significant differences in expression level of all studied proteins between OVC and control, as well as between OVC and OSCC. However, OVC showed significant lower staining scores than OSCC. Our findings demonstrate that the Akt/mTOR pathway is upregulated in OVC, indicating a role for this pathway in the development and progression of this malignancy. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  18. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    PubMed

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser(318), Ser(346), Ser(612), and Ser(789), and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R.

  19. Akt signaling leads to stem cell activation and promotes tumor development in epidermis.

    PubMed

    Segrelles, Carmen; García-Escudero, Ramón; Garín, Maria I; Aranda, Juan F; Hernández, Pilar; Ariza, José M; Santos, Mirentxu; Paramio, Jesús M; Lorz, Corina

    2014-07-01

    Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.

  20. Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action.

    PubMed

    Xu, Yingke; Nan, Di; Fan, Jiannan; Bogan, Jonathan S; Toomre, Derek

    2016-05-15

    Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.

  1. Subthalamic, not striatal, activity correlates with basal ganglia downstream activity in normal and parkinsonian monkeys

    PubMed Central

    Deffains, Marc; Iskhakova, Liliya; Katabi, Shiran; Haber, Suzanne N; Israel, Zvi; Bergman, Hagai

    2016-01-01

    The striatum and the subthalamic nucleus (STN) constitute the input stage of the basal ganglia (BG) network and together innervate BG downstream structures using GABA and glutamate, respectively. Comparison of the neuronal activity in BG input and downstream structures reveals that subthalamic, not striatal, activity fluctuations correlate with modulations in the increase/decrease discharge balance of BG downstream neurons during temporal discounting classical condition task. After induction of parkinsonism with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), abnormal low beta (8-15 Hz) spiking and local field potential (LFP) oscillations resonate across the BG network. Nevertheless, LFP beta oscillations entrain spiking activity of STN, striatal cholinergic interneurons and BG downstream structures, but do not entrain spiking activity of striatal projection neurons. Our results highlight the pivotal role of STN divergent projections in BG physiology and pathophysiology and may explain why STN is such an effective site for invasive treatment of advanced Parkinson's disease and other BG-related disorders. DOI: http://dx.doi.org/10.7554/eLife.16443.001 PMID:27552049

  2. Immunohistochemical Evaluation of AKT Protein Activation in Canine Mast Cell Tumours

    PubMed Central

    Rodriguez, S.; Fadlalla, K.; Graham, T.; Tameru, B.; Fermin, C. D.; Samuel, T.

    2011-01-01

    Summary The pathogenesis of canine mast cell tumour (MCT) remains unknown. Moreover, therapeutic options are limited and resistance to targeted drugs and recurrences are common, necessitating the identification of additional cellular targets for therapy. In this study we investigated the expression of phosphorylated AKT protein in 25 archival canine MCT samples by immunohistochemistry and examined the correlation between the immunohistochemical scores and histopathological tumour grades. AKT protein was detected in all of the samples and 24 of the 25 samples expressed the phosphorylated form of the protein, albeit with variable intensity. However, when the immunohistochemical scores of weak, intermediate and strong labelling were compared with the histopathological grades of I to III, there was no strong correlation. This study suggests that canine MCT cells have activated AKT and indicates the need for further research on the role of the AKT protein and the possibility of targeting the AKT signalling pathway in MCTs. PMID:22289273

  3. ROS-mediated Activation of AKT Induces Apoptosis Via pVHL in Prostate Cancer Cells

    PubMed Central

    Chetram, Mahandranauth A.; Bethea, Danaya A.; Jones, Kia J.; Don-Salu-Hewage, Ayesha S.; Odero-Marah, Valerie A.; Hinton, Cimona V.

    2013-01-01

    Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function. PMID:23315288

  4. Activation of Akt protects alveoli from neonatal oxygen-induced lung injury.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Coltan, Lavinia; Eaton, Farah; Barr, Amy J; Dyck, Jason R B; Thébaud, Bernard

    2011-02-01

    Bronchopulmonary dysplasia (BPD) is the main complication of extreme prematurity, resulting in part from mechanical ventilation and oxygen therapy. Currently, no specific treatment exists for BPD. BPD is characterized by an arrest in alveolar development and increased apoptosis of alveolar epithelial cells (AECs). Type 2 AECs are putative distal lung progenitor cells, capable of regenerating alveolar homeostasis after injury. We hypothesized that the protection of AEC2 death via the activation of the prosurvival Akt pathway prevents arrested alveolar development in experimental BPD. We show that the pharmacologic inhibition of the prosurvival factor Akt pathway with wortmannin during the critical period of alveolar development impairs alveolar development in newborn rats, resulting in larger and fewer alveoli, reminiscent of BPD. Conversely, in an experimental model of BPD induced by oxygen exposure of newborn rats, alveolar simplification is associated with a decreased activation of lung Akt. In vitro studies with rat lung epithelial (RLE) cells cultured in hyperoxia (95% O(2)) showed decreased apoptosis and improved cell survival after the forced expression of active Akt by adenovirus-mediated gene transfer. In vivo, adenovirus-mediated Akt gene transfer preserves alveolar architecture in the newborn rat model of hyperoxia-induced BPD. We conclude that inhibition of the prosurvival factor Akt disrupts normal lung development, whereas the expression of active Akt in experimental BPD preserves alveolar development. We speculate that the modulation of apoptosis may have therapeutic potential in lung diseases characterized by alveolar damage.

  5. Active form of AKT controls cell proliferation and response to apoptosis in hepatocellular carcinoma

    PubMed Central

    KUNTER, IMGE; ERDAL, ESRA; NART, DENIZ; YILMAZ, FUNDA; KARADEMIR, SEDAT; SAGOL, OZGUL; ATABEY, NESE

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Deregulation of the AKT signaling pathway has been found in HCC. However, the effect of AKT activation on the proliferation and apoptosis in HCC is not clear. Herein, expression of phosphorylated form of AKT (Ser 473) was investigated in HCC tumor (n=73), cirrhosis (n=17), normal liver (n=22) samples and in HCC cell lines (n=8). The results showed that expression of p-AKT was higher in tumor (53%) than in cirrhotic tissues (12%) while it was absent in normal liver (p<0.0001). p-AKT expression was also associated with number of tumor nodules and differentiation status (p<0.05). LY294002 induced cell cycle arrest at G0/G1 in SNU-449 and Mahlavu cells by decreasing expression of CDK2, CDK4, CycD1, CycD3, CycE, CycA and increasing expression of p21 and p27 as well; it also caused a decrease in the E2F1 transcriptional activity through declining phosphorylated Rb. LY294002 did not affect the basal level of apoptosis; however, it amplified cisplatin-induced apoptosis in SNU-449 cells. When the p-AKT level was decreased specifically after transfection with the DN-AKT plasmid, SNU-449 cells became more sensitive to cisplatin-induced apoptosis. HuH-7 cells with no basal p-AKT, were markedly affected by the treatment of doxorubicin. Thus, Akt signaling controls growth and chemical-induced apoptosis in HCC and p-AKT may be a potential target for therapeutic interventions in HCC patients. PMID:24337632

  6. Loss of Akt activity increases circulating soluble endoglin release in preeclampsia: identification of inter-dependency between Akt-1 and heme oxygenase-1

    PubMed Central

    Cudmore, Melissa J.; Ahmad, Shakil; Sissaoui, Samir; Ramma, Wenda; Ma, Bin; Fujisawa, Takeshi; Al-Ani, Bahjat; Wang, Keqing; Cai, Meng; Crispi, Fatima; Hewett, Peter W.; Gratacós, Eduard; Egginton, Stuart; Ahmed, Asif

    2012-01-01

    Aims Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-β (TGF-β) signalling, which is known to be elevated in preeclampsia. Methods and results Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction. PMID:21411816

  7. Potential role of AKT/mTOR signalling proteins in hairy cell leukaemia: association with BRAF/ERK activation and clinical outcome

    PubMed Central

    Lakiotaki, Eleftheria; Levidou, Georgia; Angelopoulou, Maria K.; Adamopoulos, Christos; Pangalis, Gerassimos; Rassidakis, George; Vassilakopoulos, Theodoros; Gainaru, Gabriella; Flevari, Pagona; Sachanas, Sotirios; Saetta, Angelica A.; Sepsa, Athanasia; Moschogiannis, Maria; Kalpadakis, Christina; Tsesmetzis, Nikolaos; Milionis, Vassilios; Chatziandreou, Ilenia; Thymara, Irene; Panayiotidis, Panayiotis; Dimopoulou, Maria; Plata, Eleni; Konstantopoulos, Konstantinos; Patsouris, Efstratios; Piperi, Christina; Korkolopoulou, Penelope

    2016-01-01

    The potential role of AKT/mTOR signalling proteins and its association with the Raf-MEK-ERK pathway was investigated in hairy cell leukaemia (HCL). BRAFV600E expression and activated forms of AKT, mTOR, ERK1/2, p70S6k and 4E-BP1 were immunohistochemically assessed in 77 BM biopsies of HCL patients and correlated with clinicopathological and BM microvascular characteristics, as well as with c-Caspase-3 levels in hairy cells. Additionally, we tested rapamycin treatment response of BONNA-12 wild-type cells or transfected with BRAFV600E. Most HCL cases expressed p-p70S6K and p-4E-BP1 but not p-mTOR, being accompanied by p-ERK1/2 and p-AKT. AKT/mTOR activation was evident in BONNA-12 cells irrespective of the presence of BRAFV600E mutation and was implicated in cell proliferation enhancement. In multivariate analysis p-AKT/p-mTOR/p-4E-BP1 overexpression was an adverse prognostic factor for time to next treatment conferring earlier relapse. When p-AKT, p-mTOR and p-4E-BP1 were examined separately only p-4E-BP1 remained significant. Our findings indicate that in HCL, critical proteins up- and downstream of mTOR are activated. Moreover, the strong associations with Raf-MEK-ERK signalling imply a possible biologic interaction between these pathways. Most importantly, expression of p-4E-BP1 alone or combined with p-AKT and p-mTOR is of prognostic value in patients with HCL. PMID:26893254

  8. Potential role of AKT/mTOR signalling proteins in hairy cell leukaemia: association with BRAF/ERK activation and clinical outcome.

    PubMed

    Lakiotaki, Eleftheria; Levidou, Georgia; Angelopoulou, Maria K; Adamopoulos, Christos; Pangalis, Gerassimos; Rassidakis, George; Vassilakopoulos, Theodoros; Gainaru, Gabriella; Flevari, Pagona; Sachanas, Sotirios; Saetta, Angelica A; Sepsa, Athanasia; Moschogiannis, Maria; Kalpadakis, Christina; Tsesmetzis, Nikolaos; Milionis, Vassilios; Chatziandreou, Ilenia; Thymara, Irene; Panayiotidis, Panayiotis; Dimopoulou, Maria; Plata, Eleni; Konstantopoulos, Konstantinos; Patsouris, Efstratios; Piperi, Christina; Korkolopoulou, Penelope

    2016-02-19

    The potential role of AKT/mTOR signalling proteins and its association with the Raf-MEK-ERK pathway was investigated in hairy cell leukaemia (HCL). BRAFV600E expression and activated forms of AKT, mTOR, ERK1/2, p70S6k and 4E-BP1 were immunohistochemically assessed in 77 BM biopsies of HCL patients and correlated with clinicopathological and BM microvascular characteristics, as well as with c-Caspase-3 levels in hairy cells. Additionally, we tested rapamycin treatment response of BONNA-12 wild-type cells or transfected with BRAFV600E. Most HCL cases expressed p-p70S6K and p-4E-BP1 but not p-mTOR, being accompanied by p-ERK1/2 and p-AKT. AKT/mTOR activation was evident in BONNA-12 cells irrespective of the presence of BRAFV600E mutation and was implicated in cell proliferation enhancement. In multivariate analysis p-AKT/p-mTOR/p-4E-BP1 overexpression was an adverse prognostic factor for time to next treatment conferring earlier relapse. When p-AKT, p-mTOR and p-4E-BP1 were examined separately only p-4E-BP1 remained significant. Our findings indicate that in HCL, critical proteins up- and downstream of mTOR are activated. Moreover, the strong associations with Raf-MEK-ERK signalling imply a possible biologic interaction between these pathways. Most importantly, expression of p-4E-BP1 alone or combined with p-AKT and p-mTOR is of prognostic value in patients with HCL.

  9. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation.

    PubMed

    Tessner, Teresa G; Muhale, Filipe; Riehl, Terrence E; Anant, Shrikant; Stenson, William F

    2004-12-01

    Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

  10. Activation of PI3Kγ/Akt pathway increases cardiomyocyte HMGB1 expression in diabetic environment

    PubMed Central

    Song, Jia; Liu, Qian; Tang, Han; Tao, Aibin; Wang, Hao; Kao, Raymond; Rui, Tao

    2016-01-01

    Background The high mobility group box 1 (HMGB1) protein mediates the cardiomyocyte–cardiac fibroblast interaction that contributes to induction of myocardial fibrosis in diabetes mellitus (DM). In the present study, we aim to investigate the intracellular signaling pathway that leads to cardiomyocyte HMGB1 expression under a diabetic environment. Results HMGB1 expression is increased in high concentration of glucose (HG)-conditioned cardiomyocytes. Challenging cardiomyocytes with HG also increased PI3Kγ and Akt phosphorylation. Inhibition of PI3Kγ (CRISPR/Cas9 knockout plasmid or AS605240) prevented HG-induced Akt phosphorylation and HMGB1 expression by the cardiomyocytes. In addition, inhibition of Akt (Akt1/2/3 siRNA or A6730) attenuated HG-induced HMGB1 production. Finally, challenging cardiomyocytes with HG resulted in increased reactive oxygen species (ROS) production. Treatment of cardiomyocytes with an antioxidant (Mitotempo) abolished HG-induced PI3Kγ and Akt activation, as well as HMGB1 production. Materials and Methods Isolated rat cardiomyocytes were cultured with a high concentration of glucose. Cardiomyocyte phosphatidylinositol 3-kinase gamma (PI3Kγ) and Akt activation were determined by Western blot. Cardiomyocyte HMGB1 production was evaluated with Western blot and enzyme-linked immunosorbent assay (ELISA), while cardiomyocyte oxidative stress was determined with a DCFDA fluorescence probe. Conclusions Our results suggest that the cardiomyocytes incur an oxidative stress under diabetic condition, which subsequently activates the PI3Kγ/Akt cell-signaling pathway and further increases HMGB1 expression. PMID:27821807

  11. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer.

    PubMed

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel; Teixidó, Cristina; Karachaliou, Niki; Pedersen, Martin Haar; Castellví, Josep; Garzón, Mónica; Codony-Servat, Carles; Codony-Servat, Jordi; Giménez-Capitán, Ana; Drozdowskyj, Ana; Viteri, Santiago; Larsen, Martin R; Lassen, Ulrik; Felip, Enriqueta; Bivona, Trever G; Ditzel, Henrik J; Rosell, Rafael

    2017-09-04

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.

  12. Lovastatin protects neurite degeneration in LRRK2-G2019S parkinsonism through activating the Akt/Nrf pathway and inhibiting GSK3β activity.

    PubMed

    Lin, Chin-Hsien; Lin, Han-I; Chen, Meng-Ling; Lai, Tzu-Ting; Cao, Li-Ping; Farrer, Matthew J; Wu, Ruey-Meei; Chien, Cheng-Ting

    2016-05-15

    Parkinson's disease (PD) is a progressive neurodegenerative disorder that lacks a disease-modifying therapy. Leucine-rich repeat kinase 2 (LRRK2) was implicated as the most common genetic cause of PD. We previously established a LRRK2-G2019S Drosophila model that displayed the crucial phenotypes of LRRK2 parkinsonism. Here, we used a two-step approach to identify compounds from the FDA-approved licensed drug library that could suppress neurite degeneration in LRRK2-G2019S parkinsonism. Of 640 compounds, 29 rescued neurite degeneration phenotypes and 3 restored motor disability and dopaminergic neuron loss in aged LRRK2-G2019S flies. Of these three drugs, lovastatin had the highest lipophilicity, which facilitated crossing the blood-brain barrier. In LRRK2-G2019S knock-in mice and stably transfected human dopaminergic cells, lovastatin significantly rescued neurite degeneration in a dose-dependent manner, within a range of 0.05-0.1 μm The beneficial effect of lovastatin was exerted by activating anti-apoptotic Akt/Nrf signaling and decreasing caspase 3 levels. We also observed that lovastatin inhibited GSK3β activity, a kinase downstream of Akt, by up-regulating GSK3β (Ser9) phosphorylation. This inhibition subsequently decreased tau phosphorylation, which was linked to neuronal cytoskeleton instability. Conversely, pre-treatment with the Akt inhibitor, A6730, blocked the lovastatin-induced neuroprotective effect. The rescuing effects of lovastatin in dendritic arborization of LRRK2-G2019S neurons were abolished by co-expressing either a mutant allele of Akt (Akt1(04226)) or a constitutively active form of GSK3β (sgg(S9A)). Our findings demonstrated that lovastatin restored LRRK2-G2019S neurite degeneration by augmenting Akt/NRF2 pathway and inhibiting downstream GSK3β activity, which decreased phospho-tau levels. We suggested that lovastatin is a potential disease-modifying agent for LRRK2-G2019S parkinsonism. © The Author 2016. Published by Oxford University

  13. Gab3 overexpression in human glioma mediates Akt activation and tumor cell proliferation

    PubMed Central

    Gu, Weiting; Zhang, Weifeng

    2017-01-01

    This current study tested expression and potential biological functions of Gab3 in human glioma. Gab3 mRNA and protein expression was significantly elevated in human glioma tissues and glioma cells. Its level was however low in normal brain tissues and primary human astrocytes. In both established (U251MG cell line) and primary human glioma cells, Gab3 knockdown by shRNA/siRNA significantly inhibited Akt activation and cell proliferation. Reversely, forced Gab3 overexpression in U251MG cells promoted Akt activation and cell proliferation. In vivo, the growth of U251MG tumors in nude mice was inhibited following expressing Gab3 shRNA. Akt activation in cancer tissues was also suppressed by Gab3 shRNA. Together, we conclude that Gab3 overexpression in human glioma mediates Akt activation and cancer cell proliferation. PMID:28291820

  14. δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

    PubMed

    Wang, Hong; Hong, Jungil; Yang, Chung S

    2016-11-01

    The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc.

  15. Activation of AKT by O-GlcNAcylation Induces Vascular Calcification in Diabetes

    PubMed Central

    Heath, Jack M; Sun, Yong; Yuan, Kaiyu; Bradley, Wayne E; Litovsky, Silvio; Dell’Italia, Louis J; Chatham, John C.; Wu, Hui; Chen, Yabing

    2014-01-01

    Rationale Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes. Hyperglycemia, a hallmark of diabetes, is associated with increased vascular calcification as well as increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which also was associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase (OGA) that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or OGA knockdown, promoted calcification of primary mouse vascular smooth muscle cells (VSMC). Increased O-GlcNAcylation in diabetic arteries or in the OGA knockdown VSMC upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new O-sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced VSMC calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mTOR complex 2 to AKT, and subsequently blocked Runx2 transactivity and VSMC calcification. Conclusions O-GlcNAcylation of AKT at two new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes and uncovered a key molecular mechanism underlying O-GlcNAcylation-mediated activation of AKT. PMID:24526702

  16. Constitutively expressed COX-2 in osteoblasts positively regulates Akt signal transduction via suppression of PTEN activity.

    PubMed

    Li, Ching-Ju; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling

    2011-02-01

    Cyclooxygenase-2 (COX-2) is thought to be an inducible enzyme, but increasing reports indicate that COX-2 is constitutively expressed in several organs. The status of COX-2 expression in bone and its physiological role remains undefined. Non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, which commonly suppress COX-2 activity, were reported to suppress osteoblast proliferation via Akt/FOXO3a/p27(Kip1) signaling, suggesting that COX-2 may be the key factor of the suppressive effects of NSAIDs on proliferation. Although Akt activation correlates with PTEN deficiency and cell viability, the role of COX-2 on PTEN/Akt regulation remains unclear. In this study, we hypothesized that COX-2 may be constitutively expressed in osteoblasts and regulate PTEN/Akt-related proliferation. We examined the localization and co-expression of COX-2 and p-Akt in normal mouse femurs and in cultured mouse (mOBs) and human osteoblasts (hOBs). Our results showed that osteoblasts adjacent to the trabeculae, periosteum and endosteum in mouse femurs constitutively expressed COX-2, while COX-2 co-expressed with p-Akt in osteoblasts sitting adjacent to trabeculae in vivo, and in mOBs and hOBs in vitro. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. Our findings provide

  17. Akt-dependent and Akt-independent pathways are involved in protein synthesis activation during reloading of disused soleus muscle.

    PubMed

    Mirzoev, Timur M; Tyganov, Sergey A; Shenkman, Boris S

    2017-03-01

    The purpose of our study was to assess the contribution of insulin growth factor-1-dependent and phosphatidic acid-dependent signaling pathways to activation of protein synthesis (PS) in rat soleus muscle during early recovery from unloading. Wistar rats were divided into: Control, 14HS [14-day hindlimb suspension (HS)], 3R+placebo (3-day reloading + saline administration), 3R+Wort (3-day reloading + wortmannin administration), 3R+But (3-day reloading + 1-butanol administration). SUnSET and Western blot analyses were used in this study. Wortmannin and 1-butanol induced a decrease in protein kinase B (phospho-Akt) and the rate of PS (P < 0.05) versus Control. In 3R+placebo and 3R+Wort, phosphorylation of glycogen synthase kinase 3 beta (phospho-GSK-3β) was increased versus Control (P < 0.05). Wortmannin administration during reloading did not alter phospho-p70S6K (70 kDa ribosomal protein S6 kinase) versus 3R+placebo. In 3R+But, there was a decline in phospho-GSK-3β versus 3R+placebo and Control. In 3R+But, there was a decrease in phopho-p70S6K (P < 0.05) versus 3R+placebo. These results suggest that PS activation during 3-day reloading following 14HS involves both Akt-dependent and Akt-independent pathways. Muscle Nerve 55: 393-399, 2017. © 2016 Wiley Periodicals, Inc.

  18. Molecular pharmacology and antitumor activity of PHT-427, a novel Akt/phosphatidylinositide-dependent protein kinase 1 pleckstrin homology domain inhibitor.

    PubMed

    Meuillet, Emmanuelle J; Zuohe, Song; Lemos, Robert; Ihle, Nathan; Kingston, John; Watkins, Ryan; Moses, Sylvestor A; Zhang, Shuxing; Du-Cuny, Lei; Herbst, Roy; Jacoby, Jörg J; Zhou, Li Li; Ahad, Ali M; Mash, Eugene A; Kirkpatrick, D Lynn; Powis, Garth

    2010-03-01

    Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity.

  19. Sevoflurane Postconditioning Protects Rat Hearts against Ischemia-Reperfusion Injury via the Activation of PI3K/AKT/mTOR Signaling

    PubMed Central

    Zhang, Jing; Wang, Chen; Yu, Shuchun; Luo, Zhenzhong; Chen, Yong; Liu, Qin; Hua, Fuzhou; Xu, Guohai; Yu, Peng

    2014-01-01

    Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway plays a key role in myocardial ischemia-reperfusion (I/R) injury. Mammalian target of rapamycin (mTOR), a downstream target of PI3K/AKT signaling, is necessary and sufficient to protect the heart from I/R injury. Inhaled anesthetic sevoflurane is widely used in cardiac surgeries because its induction and recovery are faster and smoother than other inhaled anesthetics. Sevoflurane proved capable of inducing postconditioning effects in the myocardium. However, the underlying molecular mechanisms for sevoflurane-induced postconditioning (SPC) were largely unclear. In the present study, we demonstrated that SPC protects myocardium from I/R injury with narrowed cardiac infarct focus, increased ATP content, and decreased cardiomyocyte apoptosis, which are mainly due to the activation of PI3K/AKT/mTOR signaling and the protection of mitochondrial energy metabolism. Application of dactolisib (BEZ235), a PI3K/mTOR dual inhibitor, abolishes the up-regulation of pho-AKT, pho-GSK, pho-mTOR, and pho-p70s6k induced by SPC, hence abrogating the anti-apoptotic effect of sevoflurane and reducing SPC-mediated protection of heart from I/R injury. As such, this study proved that PI3K/AKT/mTOR pathway plays an important role in SPC induced cardiac protection against I/R injury. PMID:25471136

  20. SiO2@antisense molecules covered by nepetalactone, extracted from Nepeta gloeocephala, inhibits ILK phosphorylation and downstream PKB/AKT signaling in HeLa cells.

    PubMed

    Dehghany Ashkezary, M; Aboee-Mehrizi, F; Moradi, P

    2017-01-01

    In this study, the anticancer property of SiO2@antisense molecules (SiO2@AMs) and SiO2@AM covered by nepetalactone (SiO2@AM/CN), extracted from Nepeta gloeocephala, was investigated. Here integrin-linked kinase (ILK) phosphorylation and protein kinase B/AKT (PKB/AKT) signaling was studied when HeLa cells were exposed to SiO2@AM and SiO2@AM/CN. First, N. gloeocephala was identified at the Iranian National Herbarium. Then, its essential oil (EO) was obtained by the hydrodistillation method. In the next step, 4aα,7α,7aα-nepetalactone was extracted from the EO, based on the spectroscopic data. To obtain SiO2@AM/CN, 1 ml of SiO2@AM was mixed with extracted nepetalactone and then strongly shaken for 30 min. Finally, serial concentrations (100, 50, 25 and 12.5 μg ml(-1)) of SiO2@AM and SiO2@AM/CN were prepared and then exposed to HeLa cells (2 × 10(5) cells per ml) for 24 h at 37 °C. After incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell-cycle analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and western blots were carried out. To find ILK phosphorylation and PKB/AKT signaling, the expression of threonine-173 (Thr-173), serine-246 (Ser-246), total ILK, AKT-Ser473, AKT-Thr308 and total AKT was investigated. HeLa cells that were treated with SiO2@AM/CN had G2/M arrest. Based on the TUNEL assay, many apoptotic cells have been shown when they were exposed to SiO2@AM/CN. Importantly, SiO2@AM/CN decreased ILK phosphorylation at Thr-173 and Ser-246 without affecting total ILK levels. Moreover, SiO2@AM/CN decreased AKT-Ser473 and AKT-Thr308 phosphorylation without affecting total PKB/AKT protein.

  1. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  2. The majority of early primordial germ cells acquire pluripotency by AKT activation.

    PubMed

    Matsui, Yasuhisa; Takehara, Asuka; Tokitake, Yuko; Ikeda, Makiko; Obara, Yuka; Morita-Fujimura, Yuiko; Kimura, Tohru; Nakano, Toru

    2014-12-01

    Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency. © 2014. Published by The Company of Biologists Ltd.

  3. Akt activation prevents the force drop induced by eccentric contractions in dystrophin-deficient skeletal muscle.

    PubMed

    Blaauw, Bert; Mammucari, Cristina; Toniolo, Luana; Agatea, Lisa; Abraham, Reimar; Sandri, Marco; Reggiani, Carlo; Schiaffino, Stefano

    2008-12-01

    Skeletal muscles of the mdx mouse, a model of Duchenne Muscular Dystrophy, show an excessive reduction in the maximal tetanic force following eccentric contractions. This specific sign of the susceptibility of dystrophin-deficient muscles to mechanical stress can be used as a quantitative test to measure the efficacy of therapeutic interventions. Using inducible transgenesis in mice, we show that when Akt activity is increased the force drop induced by eccentric contractions in mdx mice becomes similar to that of wild-type mice. This effect is not correlated with muscle hypertrophy and is not blocked by rapamycin treatment. The force drop induced by eccentric contractions is similar in skinned muscle fibers from mdx and Akt-mdx mice when stretch is applied directly to skinned fibers. However, skinned fibers isolated from mdx muscles exposed to eccentric contractions in vivo develop less isometric force than wild-type fibers and this force depression is completely prevented by Akt activation. These experiments indicate that the myofibrillar-cytoskeletal system of dystrophin-deficient muscle is highly susceptible to a damage caused by eccentric contraction when elongation is applied in vivo, and this damage can be prevented by Akt activation. Microarray and PCR analyses indicate that Akt activation induces up-regulation of genes coding for proteins associated with Z-disks and costameres, and for proteins with anti-oxidant or chaperone function. The protein levels of utrophin and dysferlin are also increased by Akt activation.

  4. Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle.

    PubMed

    Sakamoto, Kei; Arnolds, David E W; Ekberg, Ingvar; Thorell, Anders; Goodyear, Laurie J

    2004-06-25

    Activation of Akt and deactivation of GSK3 are critical signals regulating a number of cellular processes in multiple systems. Whether physical exercise alters Akt and GSK3 activity in human skeletal muscle is controversial. beta-Catenin, a GSK3 substrate and important Wnt signaling protein that alters gene transcription, has not been investigated in human skeletal muscle. In the present study, eight healthy human subjects performed 30min of cycling exercise at 75% of maximum workload (submaximal) followed by 6 bouts of 60s at 125% maximum workload (maximal). Biopsies of vastus lateralis muscle were taken at rest (basal), and within 15s following cessation of the submaximal and maximal exercise bouts. Exercise at both submaximal and maximal intensities significantly increased Akt activity (40% and 110%, respectively). Increases in Akt activity were accompanied by increases in Akt Thr(308) and Ser(473) phosphorylation, decreased GSK3alpha activity ( approximately 30% at both intensities), and increased phosphorylation of GSK3alpha Ser(21). Exercise at both intensities also decreased beta-catenin Ser(33/37)Thr(41) phosphorylation (50-60% at both intensities). These results demonstrate that Akt, GSK3, and beta-catenin signaling are regulated by exercise in human skeletal muscle, and as such identify them as possible molecular mediators of exercise's effect on metabolic and transcriptional processes in skeletal muscle.

  5. Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein.

    PubMed

    Menges, Craig W; Baglia, Laurel A; Lapoint, Randi; McCance, Dennis J

    2006-06-01

    Human papillomaviruses (HPV) are small DNA tumor viruses causally associated with cervical cancer. The early gene product E7 from high-risk HPV is considered the major transforming protein expressed by the virus. Although many functions have been described for E7 in disrupting normal cellular processes, we describe in this study a new cellular target in primary human foreskin keratinocytes (HFK), the serine/threonine kinase AKT. Expression of HPV type 16 E7 in HFK caused inhibition of differentiation, hyperproliferation, and up-regulation of AKT activity in organotypic raft cultures. The ability of E7 to up-regulate AKT activity is dependent on its ability to bind to and inactivate the retinoblastoma (Rb) gene product family of proteins. Furthermore, we show that knocking down Rb alone, with short hairpin RNAs, was sufficient to up-regulate AKT activity in differentiated keratinocytes. Up-regulation of AKT activity and loss of Rb was also observed in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Together, these data provide evidence linking inactivation of Rb by E7 in the up-regulation of AKT activity during cervical cancer progression.

  6. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  7. CARMA1 is required for Akt-mediated NF-kappaB activation in T cells.

    PubMed

    Narayan, Preeti; Holt, Brittany; Tosti, Richard; Kane, Lawrence P

    2006-03-01

    Many details of the generic pathway for induction of NF-kappaB have been delineated, but it is still not clear how multiple, diverse receptor systems are able to converge on this evolutionarily conserved family of transcription factors. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for coupling the common elements of the NF-kappaB pathway to the T-cell receptor (TCR) and CD28. We previously demonstrated a role for the serine/threonine kinase Akt in CD28-mediated NF-kappaB induction. Using a CARMA1-deficient T-cell line, we have now found that the CARMA complex is required for induction of NF-kappaB by Akt, in cooperation with protein kinase C activation. Furthermore, using a novel selective inhibitor of Akt, we confirm that Akt plays a modulatory role in NF-kappaB induction by the TCR and CD28. Finally, we provide evidence for a physical and functional interaction between Akt and CARMA and for Akt-dependent phosphorylation of Bcl10. Therefore, in T cells, Akt impinges upon NF-kappaB signaling through at least two separate mechanisms.

  8. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    DTIC Science & Technology

    2009-03-01

    prenylation signal derived from K- Ras rendered Akt1 catalytically inactive when fused to the N-terminal PH-domain (Fig. 1). Fusion of this motif to...were treated with either cyclo- dextrin (CD) or water-soluble cholesterol ( Chol ) or with CD followed by cholesterol treatment (CD Chol ). Cells

  9. Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

    PubMed Central

    Ocana, Alberto; Vera-Badillo, Francisco; Al-Mubarak, Mustafa; Templeton, Arnoud J.; Corrales-Sanchez, Verónica; Diez-Gonzalez, Laura; Cuenca-Lopez, María D.; Seruga, Bostjan; Pandiella, Atanasio; Amir, Eitan

    2014-01-01

    Background Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear. Methods PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model. Results Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13). Conclusion Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway. PMID:24777052

  10. Secreted protein acidic, rich in cysteine (SPARC), mediates cellular survival of gliomas through AKT activation.

    PubMed

    Shi, Qing; Bao, Shideng; Maxwell, Jill A; Reese, Elizabeth D; Friedman, Henry S; Bigner, Darell D; Wang, Xiao-Fan; Rich, Jeremy N

    2004-12-10

    Secreted protein acidic, rich in cysteine (SPARC), is an extracellular matrix protein expressed in many advanced cancers, including malignant gliomas. We and others have previously shown that human glioma cell lines engineered to overexpress SPARC adopt an invasive phenotype. We now show that SPARC expression increases cell survival under stress initiated by serum withdrawal through a decrease in apoptosis. Phosphatidylinositol 3-OH kinase/AKT is a potent pro-survival pathway that contributes to the malignancy of gliomas. Cells expressing SPARC display increased AKT activation with decreased caspase 3/7 activity. Exogenous SPARC rapidly induces AKT phosphorylation, an effect that is blocked by a neutralizing SPARC antibody. Furthermore, AKT activation is essential for the anti-apoptotic effects of SPARC as the decreased apoptosis and caspase activity associated with SPARC expression can be blocked with dominant-negative AKT or a specific AKT inhibitor. As tumor cells face stressful microenvironments particularly during the process of invasion, these results suggest that SPARC functions, in part, to promote tumor progression by enabling tumor cells to survive under stressful conditions.

  11. ADP-ribosylation factor-like GTPase 15 enhances insulin-induced AKT phosphorylation in the IR/IRS1/AKT pathway by interacting with ASAP2 and regulating PDPK1 activity.

    PubMed

    Zhao, Jie; Wang, Min; Deng, Wuquan; Zhong, Daping; Jiang, Youzhao; Liao, Yong; Chen, Bing; Zhang, Xiaoli

    2017-05-13

    Decreased phosphorylation in the insulin signalling pathway is a hallmark of insulin resistance. The causes of this phenomenon are complicated and multifactorial. Recently, genomic analyses have identified ARL15 as a new candidate gene related to diabetes. However, the ARL15 protein function remains unclear. Here, we show that ARL15 is upregulated by insulin stimulation. This effect was impaired in insulin-resistant pathophysiology in TNF-α-treated C2C12 myotubes and in the skeletal muscles of leptin knockout mice. In addition, ARL15 localized to the cytoplasm in the resting state and accumulated in the Golgi apparatus around the nucleus upon insulin stimulation. ARL15 overexpression can enhance the phosphorylation of the key insulin signalling pathway molecules IR, IRS1 and AKT in C2C12 myotubes. Moreover, ARL15 knockdown can also specifically inhibit the phosphorylation of PDPK1 Ser241, thereby reducing PDPK1 activity and its downstream phosphorylation of AKT Thr308. Co-immunoprecipitation assays identified ASAP2 as an ARL15-interacting protein. In conclusion, we have identified that ARL15 acts as an insulin-sensitizing effector molecule to upregulate the phosphorylation of members of the canonical IR/IRS1/PDPK1/AKT insulin pathway by interacting with its GAP ASAP2 and activating PDPK1. This research may provide new insights into GTPase-mediated insulin signalling regulation and facilitate the development of new pharmacotherapeutic targets for insulin sensitization. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The Akt signaling pathway

    PubMed Central

    Madhunapantula, SubbaRao V; Mosca, Paul J

    2011-01-01

    Studies using cultured melanoma cells and patient tumor biopsies have demonstrated deregulated PI3 kinase-Akt3 pathway activity in ∼70% of melanomas. Furthermore, targeting Akt3 and downstream PRAS40 has been shown to inhibit melanoma tumor development in mice. Although these preclinical studies and several other reports using small interfering RNAs and pharmacological agents targeting key members of this pathway have been shown to retard melanoma development, analysis of early Phase I and Phase II clinical trials using pharmacological agents to target this pathway demonstrate the need for (1) selection of patients whose tumors have PI3 kinase-Akt pathway deregulation, (2) further optimization of therapeutic agents for increased potency and reduced toxicity, (3) the identification of additional targets in the same pathway or in other signaling cascades that synergistically inhibit the growth and progression of melanoma, and (4) better methods for targeted delivery of pharmaceutical agents inhibiting this pathway. In this review we discuss key potential targets in PI3K-Akt3 signaling, the status of pharmacological agents targeting these proteins, drugs under clinical development, and strategies to improve the efficacy of therapeutic agents targeting this pathway. PMID:22157148

  13. Phase I pharmacokinetic and pharmacodynamic study of triciribine phosphate monohydrate, a small-molecule inhibitor of AKT phosphorylation, in adult subjects with solid tumors containing activated AKT.

    PubMed

    Garrett, Christopher R; Coppola, Domenico; Wenham, Robert M; Cubitt, Christopher L; Neuger, Anthony M; Frost, Timothy J; Lush, Richard M; Sullivan, Daniel M; Cheng, Jin Q; Sebti, Saïd M

    2011-12-01

    Triciribine phosphate is a potent, small-molecule inhibitor of activation of all three isoforms of AKT in vitro. AKT is an intracellular protein that, when activated, leads to cellular division; it is dysregulated in a large number of malignancies, and constitutively activating AKT mutations are present in a minority of cancers. In this phase I study triciribine phosphate monohydrate (TCN-PM) was administered to subjects whose tumors displayed evidence of increased AKT phosphorylation (p-AKT) as measured by immunohistochemical analysis (IHC). TCN-PM was administered over 30 min on days 1, 8 and 15 of a 28-day cycle. Tumor biopsy specimens, collected before treatment and on day +15, were assessed for p-AKT by IHC and western blot analyses. Nineteen subjects were enrolled; 13 received at least one cycle of therapy, and a total of 34 complete cycles were delivered. One subject was treated at the 45 mg/m(2) dose before the study was closed due to its primary objective having been met. No dose-limiting toxic effects were observed. Modest decreases in tumor p-AKT following therapy with TCN-PM were observed at the 35 mg/m(2) and 45 mg/m(2) dose levels, although definitive conclusions were limited by the small sample size. These preliminary data suggest that treatment with TCN-PM inhibits tumor p-AKT at doses that were tolerable. Although single agent activity was not observed in this enriched population, further combination studies of TCN-PM with other signal transduction pathway inhibitors in solid tumors is warranted.

  14. Phase I pharmacokinetic and pharmacodynamic study of triciribine phosphate monohydrate, a small-molecule inhibitor of AKT phosphorylation, in adult subjects with solid tumors containing activated AKT

    PubMed Central

    Garrett, Christopher R.; Coppola, Domenico; Wenham, Robert M.; Cubitt, Christopher L.; Neuger, Anthony M.; Frost, Timothy J.; Lush, Richard M.; Sullivan, Daniel M.; Cheng, Jin Q.; Sebti, Saïd M.

    2015-01-01

    Summary Purpose Triciribine phosphate is a potent, small-molecule inhibitor of activation of all three isoforms of AKT in vitro. AKT is an intracellular protein that, when activated, leads to cellular division; it is dysregulated in a large number of malignancies, and constitutively activating AKT mutations are present in a minority of cancers. Patients and methods In this phase I study triciribine phosphate monohydrate (TCN-PM) was administered to subjects whose tumors displayed evidence of increased AKT phosphorylation (p-AKT) as measured by immunohistochemical analysis (IHC). TCN-PM was administered over 30 min on days 1, 8 and 15 of a 28-day cycle. Tumor biopsy specimens, collected before treatment and on day +15, were assessed for p-AKT by IHC and western blot analyses. Results Nineteen subjects were enrolled; 13 received at least one cycle of therapy, and a total of 34 complete cycles were delivered. One subject was treated at the 45 mg/m2 dose before the study was closed due to its primary objective having been met. No dose-limiting toxic effects were observed. Modest decreases in tumor p-AKT following therapy with TCN-PM were observed at the 35 mg/m2 and 45 mg/m2 dose levels, although definitive conclusions were limited by the small sample size. Conclusions These preliminary data suggest that treatment with TCN-PM inhibits tumor p-AKT at doses that were tolerable. Although single agent activity was not observed in this enriched population, further combination studies of TCN-PM with other signal transduction pathway inhibitors in solid tumors is warranted. PMID:20644979

  15. Loss of Mel-18 induces tumor angiogenesis through enhancing the activity and expression of HIF-1α mediated by the PTEN/PI3K/Akt pathway.

    PubMed

    Park, J H; Lee, J Y; Shin, D H; Jang, K S; Kim, H J; Kong, Gu

    2011-11-10

    Mel-18 has been implicated in several processes in tumor progression, in which the Akt pathway is involved as an important key molecular event. However, the function of Mel-18 in human cancers has not been fully established yet. Here, we examined the effect of Mel-18 on tumor angiogenesis in human breast cancer, and found that Mel-18 was a novel regulator of HIF-1α. Mel-18 negatively regulated the HIF-1α expression and its target gene VEGF transcription during both normoxia and hypoxia. We demonstrated that Mel-18 regulated the HIF-1α expression and activity via the PI3K/Akt pathway. Loss of Mel-18 downregulated Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression, consequently activating the PI3K/Akt/MDM2 pathway, and leading to an increase of HIF-1α protein level. Mel-18 modulated the HIF-1α transcriptional activity via regulating the cytoplasmic retention of FOXO3a, a downstream effector of Akt, and recruitment of HIF-1α/CBP complex to the VEGF promoter. Furthermore, our data shows that Mel-18 blocked tumor angiogenesis both in vitro and in vivo. Mel-18 overexpression inhibited in vitro tube formation in human umbilical endothelial cells (HUVECs). Xenografts in NOD/SCID mice derived from stably Mel-18 knocked down MCF7 human breast cancer cells showed increased tumor volume, microvessel density, and phospho-Akt and HIF-1α expression levels. In conclusion, our findings provide that Mel-18 is a novel regulator of tumor angiogenesis through regulating HIF-1α and its target VEGF expressions mediated by the PTEN/PI3K/Akt pathway, suggesting a new tumor-suppressive role of Mel-18 in human breast cancer.

  16. NEDD4 ubiquitin ligase is a putative oncogene in endometrial cancer that activates IGF-1R/PI3K/Akt signaling.

    PubMed

    Zhang, Yuping; Goodfellow, Renee; Li, Yujun; Yang, Shujie; Winters, Christopher J; Thiel, Kristina W; Leslie, Kimberly K; Yang, Baoli

    2015-10-01

    The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression

    PubMed Central

    Henderson, Veronica; Smith, Basil; Burton, Liza J; Randle, Diandra; Morris, Marisha; Odero-Marah, Valerie A

    2015-01-01

    Snail, a zinc-finger transcription factor, induces epithelial-mesenchymal transition (EMT), which is associated with increased cell migration and metastasis in cancer cells. Rac1 is a small G-protein which upon activation results in formation of lamellipodia, the first protrusions formed by migrating cells. We have previously shown that Snail promotes cell migration through down-regulation of maspin tumor suppressor. We hypothesized that Snail's regulation of cell migration may also involve Rac1 signaling regulated by PI3K/AKT and/or MAPK pathways. We found that Snail overexpression in LNCaP and 22Rv1 prostate cancer cells increased Rac1 activity associated with increased cell migration, and the Rac1 inhibitor, NSC23766, could inhibit Snail-mediated cell migration. Conversely, Snail downregulation using shRNA in the aggressive C4–2 prostate cancer cells decreased Rac1 activity and cell migration. Moreover, Snail overexpression increased ERK and PI3K/AKT activity in 22Rv1 prostate cancer cells. Treatment of Snail-overexpressing 22Rv1 cells with LY294002, PI3K/AKT inhibitor or U0126, MEK inhibitor, decreased cell migration significantly, but only LY294002 significantly reduced Rac1 activity, suggesting that Snail promotes Rac1 activation via the PI3K/AKT pathway. Furthermore, 22Rv1 cells overexpressing Snail displayed decreased maspin levels, while inhibition of maspin expression in 22Rv1 cells with siRNA, led to increased PI3K/AKT, Rac1 activity and cell migration, without affecting ERK activity, suggesting that maspin is upstream of PI3K/AKT. Overall, we have dissected signaling pathways by which Snail may promote cell migration through MAPK signaling or alternatively through PI3K/AKT-Rac1 signaling that involves Snail inhibition of maspin tumor suppressor. This may contribute to prostate cancer progression. PMID:26207671

  18. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    PubMed

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  19. Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

    PubMed

    Avota, E; Avots, A; Niewiesk, S; Kane, L P; Bommhardt, U; ter Meulen, V; Schneider-Schaulies, S

    2001-06-01

    Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

  20. Angiotensin type I receptor blockade in conjunction with enhanced Akt activation restores coronary collateral growth in the metabolic syndrome

    PubMed Central

    Jadhav, Rashmi; Dodd, Tracy; Smith, Erika; Bailey, Erin; DeLucia, Angelo L.; Russell, James C.; Madison, Rowan; Potter, Barry; Walsh, Kenneth; Jo, Hanjoong

    2011-01-01

    We have previously demonstrated that Akt was required for repetitive ischemia (RI)-induced coronary collateral growth (CCG) in healthy rats but was not activated by RI in the metabolic syndrome (JCR:LA-cp rats) where CCG was impaired. Here we hypothesized that failure of angiotensin type I receptor (AT1R) blockers to restore Akt activation is a key determinant of their inability to completely restore CCG in the metabolic syndrome. Therefore, we investigated whether adenovirus-mediated delivery of constitutively active Akt (MyrAkt-Adv) in conjunction with AT1R blockade (candesartan) was able to restore RI-induced CCG in JCR:LA-cp rats. Successful myocardial MyrAkt-Adv delivery was confirmed by a >80% transduction efficiency and an approximately fourfold increase in Akt expression and activation. CCG was assessed by myocardial blood flow measurements in the normal and collateral-dependent zones. MyrAkt-Adv alone significantly increased RI-induced CCG in JCR:LA-cp rats (∼30%), but it completely restored CCG in conjunction with administration of candesartan. In contrast, dominant negative Akt (DN-Akt-Adv) reversed the beneficial effect of candesartan on CCG in JCR:LA-cp rats. We conclude that optimal restoration of coronary collateral growth in JCR:LA-cp rats requires a combination of AT1R blockade with constitutive Akt activation. These findings may carry implications for metabolic syndrome patients in need of coronary revascularization. PMID:21335466

  1. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

    PubMed

    Costa, Céu; Pereira, Sofia; Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A; Ferreira, José Alexandre; Santos, Lúcio L

    2015-01-01

    Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

  2. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics

    PubMed Central

    Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M.; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A.; Ferreira, José Alexandre; Santos, Lúcio L.

    2015-01-01

    Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

  3. An active IGF-1R-AKT signaling imparts functional heterogeneity in ovarian CSC population.

    PubMed

    Singh, Ram K; Dhadve, Ajit; Sakpal, Asmita; De, Abhijit; Ray, Pritha

    2016-11-07

    Deregulated IGF-1R-AKT signaling influences multiple nodes of cancer cell physiology and assists in migration, metastasis and acquirement of radio/chemoresistance. Enrichment of cancer stem cells (CSC) positively correlates with radio/chemoresistance development in various malignancies. It is unclear though, how IGF-1R-AKT signalling shapes CSC functionality especially in ovarian cancer. Previously we showed that upregulated IGF-1R expression is essential to initiate platinum-taxol resistance at early stage which declines with elevated levels of activated AKT at late resistant stage in ovarian cancer cells. Here, we investigated the effect of this oscillatory IGF-1R-AKT signalling upon CSC functionality during generation of chemoresistance. While gradual increase in CSC properties from early (ER) to late (LR) resistant stages was observed in three different (cisplatin/paclitaxel/cisplatin-paclitaxel) cellular models created in two ovarian cancer cell lines, the stemness gene expressions (oct4/sox2/nanog) reached a plateau at early resistant stages. Inhibition of IGF-1R only at ER and AKT inhibition only at LR stages significantly abrogated the CSC phenotype. Interestingly, real time bioluminescence imaging showed CSCs of ER stages possessed faster tumorigenic potential than CSCs belonging to LR stages. Together, our data suggest that IGF-1R-AKT signalling imparts functional heterogeneity in CSCs during acquirement of chemoresistance in ovarian carcinoma.

  4. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress.

    PubMed

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H2O2) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H2O2-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H2O2 were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H2O2. Reversely, escin was more potent against H2O2 damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H2O2 was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

    PubMed

    Cherian, Mathew A; Baydoun, Hicham H; Al-Saleem, Jacob; Shkriabai, Nikoloz; Kvaratskhelia, Mamuka; Green, Patrick; Ratner, Lee

    2015-10-23

    Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

  6. Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells.

    PubMed

    Lu, Jing; Caplan, Michael S; Li, Dan; Jilling, Tamas

    2008-05-01

    We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.

  7. Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells

    PubMed Central

    Lu, Jing; Caplan, Michael S.; Li, Dan; Jilling, Tamas

    2009-01-01

    We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation. PMID:18356536

  8. Impact of the PI3-kinase/Akt pathway on ITAM and hemITAM receptors: haemostasis, platelet activation and antithrombotic therapy.

    PubMed

    Moroi, Alyssa J; Watson, Steve P

    2015-04-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated in response to various stimulants, and they regulate many processes including inflammation; the stress response; gene transcription; and cell proliferation, differentiation, and death. Increasing reports have shown that the PI3Ks and their downstream effector Akt are activated by several platelet receptors that regulate platelet activation and haemostasis. Platelets express two immunoreceptor tyrosine based activation motif (ITAM) receptors, collagen receptor glycoprotein VI (GPVI) and Fcγ receptor IIA (FcγRIIA), which are characterized by two YxxL sequences separated by 6-12 amino acids. Activation of an ITAM receptor initiates a reaction cascade via its YxxL sequence in which signaling molecules such as spleen tyrosine kinase (Syk), linker for activation of T cells (LAT) and phospholipase C γ2 (PLCγ2) become activated, leading to platelet activation. Platelets also express another receptor, C-type lectin 2 (CLEC-2), which has a single YxxL sequence, so it is appropriately called a hemITAM receptor. ITAM receptors and the hemITAM receptor share many signaling features. Here we will summarize our current knowledge about how the PI3K/Akt pathway regulates (hem)ITAM receptor-mediated platelet activation and haemostasis and discuss the possible benefits of targeting PI3K/Akt as an antithrombotic therapy.

  9. PI3K-Independent AKT Activation in Cancers: A Treasure Trove for Novel Therapeutics

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2012-01-01

    AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors. PMID:22307544

  10. Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

    SciTech Connect

    Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

    2008-05-01

    Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60 min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.

  11. TC21 mediates transformation and cell survival via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB signaling pathway.

    PubMed

    Rong, Rong; He, Qin; Liu, Yusen; Sheikh, M Saeed; Huang, Ying

    2002-02-07

    The signaling pathways of TC21-mediated transformation and cell survival are not well-established. In this study, we have investigated the role of PI3-K/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. We found that oncogenic-TC21 stimulated the PI3-K activity. This was associated with the activation of Akt, a key component of PI3-K signaling pathway. We also found that TC21 interacted and formed complex with PI3-K. Mutations in the GTP-binding region of TC21, which enhanced GTP-binding potential of this protein, also stimulated its association with PI3-K, suggesting that PI3-K may preferentially interact with the GTP-bound form. Suppression of PI3-K and Akt by specific inhibitors LY294002 and Wortmannin reversed TC21-induced transformation. Likewise, inhibition of PI3-K activity by the PI3-K phosphotase PTEN reduced TC21-mediated focus formation in NIH3T3 cells. Investigation of TC21's effect on cell survival revealed that mutant-TC21 expressing cells were more resistant to etoposide- and cisplatin-induced cell death, and this was associated with the activation of anti-apoptotic protein NF-kappaB, a downstream target of Akt. Treatment of PI3-K inhibitor LY294002 significantly suppressed TC21-mediated NF-kappaB activation. In conclusion, we have identified PI3-K as an effector of TC21 and demonstrated that the PI3-K/Akt signaling pathway plays important roles in TC21-mediated transformation and cell survival.

  12. ANDROGEN RECEPTOR LEVELS ARE UPREGULATED BY AKT IN PROSTATE CANCER

    PubMed Central

    Ha, Susan; Ruoff, Rachel; Kahoud, Nicole; Franke, Thomas F.; Logan, Susan K.

    2013-01-01

    Multiple lines of evidence suggest a functional link between the androgen receptor (AR) and the serine/threonine kinase Akt in the development and progression of prostate cancer. To investigate the impact of Akt activity on AR homeostasis, we treated androgen-dependent LNCaP and LAPC-4 prostate cancer cells with Akt inhibitor. Akt inhibition decreased AR expression, suggesting that Akt activity was required for regulation of AR protein levels. However, while androgen-independent LNCaP-abl cells also showed diminished AR protein levels in response to Akt inhibition, treatment of androgen-independent LNCaP-AI cells failed to alter AR protein levels upon similar treatment, suggesting that AR protein levels in these androgen-independent prostate cells were regulated by mechanisms independent of Akt activation. Regulation of AR, downstream of activated Akt, also was observed in vivo when examining transgenic mice that overexpress constitutively active mutant myristoylated (myr)-Akt1 in the prostate. Transgenic mice animals expressing activated myr-Akt1 exhibited higher levels of AR mRNA and protein. Expression of activated myr-Akt1 did not alter prostate cell growth and no significant size differences between prostate tissues derived from transgenic animals were observed when comparing transgenic to wild-type mice. Still, transgenic mice overexpressing Akt exhibited higher levels of γH2AX and phosphorylated Chk2 in prostate tissue. These changes in markers associated with oncogene-induced senescence confirmed significant altered signaling in the transgenic mouse model. Overall, results presented here suggest that AR levels are regulated by the Akt pathway. PMID:21317204

  13. Germline activating AKT3 mutation associated with megalencephaly, polymicrogyria, epilepsy and hypoglycemia.

    PubMed

    Nellist, Mark; Schot, Rachel; Hoogeveen-Westerveld, Marianne; Neuteboom, Rinze F; van der Louw, Elles J T M; Lequin, Maarten H; Bindels-de Heus, Karen; Sibbles, Barbara J; de Coo, René; Brooks, Alice; Mancini, Grazia M S

    2015-03-01

    Activating germ-line and somatic mutations in AKT3 (OMIM 611223) are associated with megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH; OMIM # 615937) and megalencephaly-capillary malformation (MCAP; OMIM # 602501). Here we report an individual with megalencephaly, polymicrogyria, refractory epilepsy, hypoglycemia and a germline AKT3 mutation. At birth, head circumference was 43 cm (5 standard deviations above the mean). No organomegaly was present, but there was generalized hypotonia, joint and skin laxity, developmental delay and failure to thrive. At 6 months of age the patient developed infantile spasms that were resistant to antiepileptic polytherapy. Recurrent hypoglycemia was noted during treatment with adrenocorticotropic hormone but stabilized upon introduction of continuous, enriched feeding. The infantile spasms responded to the introduction of a ketogenic diet, but the hypoglycemia recurred until the diet was adjusted for increased resting energy expenditure. A novel, de novo AKT3 missense variant (exon 5; c.548T>A, p.(V183D)) was identified and shown to activate AKT3 by in vitro functional testing. We hypothesize that the sustained hypoglycemia in this patient is caused by increased glucose utilization due to activation of AKT3 signaling. This might explain the efficacy of the ketogenic diet in this individual.

  14. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling.

    PubMed

    Han, Yong-Seok; Lee, Jun Hee; Lee, Sang Hun

    2015-05-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

  15. Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death.

    PubMed

    Lee, Gilbert Aaron; Lai, Yein-Gei; Chen, Ray-Jade; Liao, Nan-Shih

    2017-04-01

    Astrocytes play a pivotal role in neuronal survival under the condition of post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. IL-15 supports survival of multiple lymphocyte lineages in the peripheral immune system, but the role of IL-15 in inflammatory disease of the central nervous system is not well defined. Recent research has shown an increase of IL-15-expressing astrocytes in the ischemic brain. Since astrocytes promote neuron survival under cerebral ischemia by buffering excess extracellular glutamate and producing growth factors, recovery of astrocyte function could be of benefit for stroke therapy. Here, we report that IL-15 is the pro-survival cytokine that prevents astrocyte death from oxygen glucose deprivation (OGD)-induced damage. Astrocytes up-regulate expression of the IL-15/IL-15Rα complex under OGD, whereas OGD down-regulates the levels of pSTAT5 and pAkt in astrocytes. IL-15 treatment ameliorates the decline of pAkt, decreases the percentage of annexin V(+) cells, inhibits the activation of caspase-3, and activates the Akt pathway to promote astrocyte survival in response to OGD. We further identified that activation of Akt, but not PKCα/βI, is essential for astrocyte survival under OGD. Taken together, this study reveals the function of IL-15 in astrocyte survival via Akt phosphorylation in response to OGD-induced damage.

  16. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  17. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    PubMed

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

  18. Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy

    PubMed Central

    Moc, Courtney; Taylor, Amy E.; Chesini, Gino P.; Zambrano, Cristina M.; Barlow, Melissa S.; Zhang, Xiaoxue; Gustafsson, Åsa B.; Purcell, Nicole H.

    2015-01-01

    Aims To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. Methods and results To investigate the in vivo requirement for ‘physiological’ control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Conclusion Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. PMID:25411382

  19. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    PubMed

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer.

  20. Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

    PubMed Central

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-01-01

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt. PMID:25228694

  1. Subcutaneous adipocytes promote melanoma cell growth by activating the Akt signaling pathway: role of palmitic acid.

    PubMed

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-10-31

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.

  2. The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

    PubMed Central

    Zhang, Yun-Hui; Zhang, Jin; Song, Jian-Nan; Xu, Xue; Cai, Jin-Song; Zhou, Yang; Gao, Jin-Gui

    2016-01-01

    We investigated roles of PI3K-AKT-mTOR pathway in recovery from general anesthesia. Sprague-Dawley rats divided into five groups: saline+artificial cerebrospinal fluid (ACSF; Group A), ketamine+ACSF (Group B), ketamine+IGF-1 (Group C), ketamine+PI3K inhibitor (Group D), and PI3K/Akt agonists (Group E). Proportion of δ waves on ECoGs was recorded. Rats were tested for duration of loss of righting reflex (LORR), ataxic period and behavior in Morris water maze. mRNA and protein expression of members of PI3K-AKT-mTOR pathway were measured by RT-qPCR and Western blots. Histopathologic changes in hippocampal tissues observed by HE staining. We found that the proportion of δ waves decreased in Group C, while increased in Group D compared with Group B; the durations of LORR and ataxic period were shorter in Group C, but longer in Group D. In Morris water maze, escape latency (EL) and duration and frequency of staying on platform was shorter in Group C and longer in Group D than in Group B. Group A exhibited low expression of proteins in PI3K-AKT-mTOR pathway, while p-AKT, p-mTOR and p-P70S6K expression increased in cerebral cortex, brain stem, and thalamus in Group C. By contrast, expression of those proteins was lower in Group D than Group B. Those proteins expressions were higher in Group E than in Group A. HE staining showed that anesthesia may induce cell apoptosis in rat hippocampal CA1 areas, and PI3K/Akt agonists could inhibit apoptosis. Our results suggest that activation of PI3K-AKT-mTOR pathway may promote recovery from general anesthesia and enhance spatial learning and memory. PMID:27340771

  3. Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition.

    PubMed

    Fan, Yongjun; Dickman, Kathleen G; Zong, Wei-Xing

    2010-03-05

    The high glucose consumption of tumor cells even in an oxygen-rich environment, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncoproteins such as Akt and c-Myc regulate cell metabolism. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arising from different oncogenic activities. Here, we established a dual-regulatable FL5.12 pre-B cell line in which myristoylated Akt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen receptor, is activated by 4-hydroxytamoxifen. Using this system, we directly compared the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis, whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, although both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs.

  4. Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling

    PubMed Central

    Miyazaki, Mitsunori; McCarthy, John J; Fedele, Mark J; Esser, Karyn A

    2011-01-01

    Abstract The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 β, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2. PMID:21300751

  5. Activation of PI3K-Akt-GSK3{beta} pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

    SciTech Connect

    Gong Rujun . E-mail: rgong@Brown.edu; Rifai, Abdalla; Dworkin, Lance D.

    2005-04-29

    Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-{alpha}-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-{alpha}-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3{beta} or an uninhibitable mutant GSK3{beta}, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3{beta}) in HKC. Overexpression of wild type GSK3{beta} did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3{beta} abolished HGF inhibition of basal and TNF-{alpha} stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3{beta} are required for HGF-induced suppression of RANTES in HKC.

  6. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

    PubMed

    Tanaka, Yuichi; Gavrielides, M Veronica; Mitsuuchi, Yasuhiro; Fujii, Teruhiko; Kazanietz, Marcelo G

    2003-09-05

    Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells

  7. Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice

    PubMed Central

    Shioi, Tetsuo; McMullen, Julie R.; Kang, Peter M.; Douglas, Pamela S.; Obata, Toshiyuki; Franke, Thomas F.; Cantley, Lewis C.; Izumo, Seigo

    2002-01-01

    One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth. PMID:11909972

  8. Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

    PubMed Central

    Kontos, Christopher D.; Stauffer, Thomas P.; Yang, Wen-Pin; York, John D.; Huang, Liwen; Blanar, Michael A.; Meyer, Tobias; Peters, Kevin G.

    1998-01-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival. PMID:9632797

  9. Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt.

    PubMed

    Kontos, C D; Stauffer, T P; Yang, W P; York, J D; Huang, L; Blanar, M A; Meyer, T; Peters, K G

    1998-07-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

  10. High constitutive Akt2 activity in U937 promonocytes: effective reduction of Akt2 phosphorylation by the histamine H2-receptor and the β2-adrenergic receptor.

    PubMed

    Werner, Kristin; Neumann, Detlef; Seifert, Roland

    2016-01-01

    Histamine (HA) is approved for the treatment of acute myeloid leukemia (AML). Its antileukemic activity is related to histamine H2-receptor (H2R)-mediated inhibition of reactive oxygen species (ROS) production in myeloid cells facilitating survival of antineoplastic lymphocytes. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which plays a crucial role in cell survival and proliferation, is constitutively activated in leukemic cells of most AML patients resulting in poor survival prognosis. In a proof-of-principle experiment using a human phosphorylated mitogen-activated protein kinase (MAPK) array, we found high phosphorylation levels of Akt2 in U937 promonocytes that was abrogated by HA or selective H2R agonists. The H2R and the β2-adrenergic receptor (β2AR) are Gs-protein-coupled receptors. Stimulation results in adenylyl cyclase activation followed by generation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). In our present study, we evaluated the pharmacological profile of the H2R and the β2AR regarding Akt2 phosphorylation at Ser474 via western blot analysis and ELISA and cAMP accumulation via HPLC-MS/MS in U937 promonocytes. H2R and β2AR agonists concentration-dependently decreased Akt2 phosphorylation at Ser474. Deviations of potencies and efficacies of agonists in Akt2 phosphorylation and cAMP accumulation assays indicated participation of cAMP-independent signaling in GPCR-induced reduction of Akt2 phosphorylation. Accordingly, our study supports the concept of functional selectivity of the H2R and the β2AR in U937 promonocytes. In summary, we extended the antileukemic mechanism of HA via H2R and revealed the potential of β2AR agonists, which are already approved in the treatment of bronchial asthma and chronic obstructive pulmonary disease, as antileukemic drugs.

  11. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  12. Sustained Activation of Akt Elicits Mitochondrial Dysfunction to Block Plasmodium falciparum Infection in the Mosquito Host

    PubMed Central

    Drexler, Anna L.; Antonova-Koch, Yevgeniya; Sakaguchi, Danielle; Napoli, Eleonora; Wong, Sarah; Price, Mark S.; Eigenheer, Richard; Phinney, Brett S.; Pakpour, Nazzy; Pietri, Jose E.; Cheung, Kong; Georgis, Martha; Riehle, Michael

    2013-01-01

    The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3–5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the

  13. Resveratrol rescues hyperglycemia-induced endothelial dysfunction via activation of Akt

    PubMed Central

    Li, Jin-yi; Huang, Wei-qiang; Tu, Rong-hui; Zhong, Guo-qiang; Luo, Bei-bei; He, Yan

    2017-01-01

    Resveratrol (RSV), a phytoalexin, has shown to prevent endothelial dysfunction and reduce diabetic vascular complications and the risk of cardiovascular diseases. The aim of this study was to investigate the signaling mechanisms underlying the protecting effects of RSV against endothelial dysfunction during hyperglycemia in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with RSV, and then exposed to high glucose (HG, 30 mmol/L). Akt-Ser473 phosphorylation, eNOS-Ser1177 phosphorylation, and PTEN protein levels in the cells were detected using Western blot. For in vivo studies, WT and Akt−/− mice were fed a normal diet containing RSV (400 mg·kg−1·d−1) for 2 weeks, then followed by injection of STZ to induce hyperglycemia (300 mg/dL). Endothelial function was evaluated using aortic rings by assessing ACh-induced vasorelaxation. RSV (5–20 μmol/L) dose-dependently increased Akt-Ser473 phosphorylation, accompanied by increased eNOS-Ser1177 phosphorylation in HUVECs; these effects were more prominent under HG stimulation. Transfection with Akt siRNA abolished RSV-enhanced eNOS phosphorylation and NO release. Furthermore, RSV (5–20 μmol/L) dose-dependently decreased the levels of PTEN, which was significantly increased under HG stimulation, and PTEN overexpression abolished RSV-stimulated Akt phosphorylation in HG-treated HUVECs. Moreover, RSV dramatically increased 26S proteasome activity, which induced degradation of PTEN. In in vivo studies, pretreatment with RSV significantly increased Akt and eNOS phosphorylation in aortic tissues and ACh-induced vasorelaxation, and improved diabetes-induced endothelial dysfunction in wild-type mice but not in Akt−/− mice. RSV attenuates endothelial function during hyperglycemia via activating proteasome-dependent degradation of PTEN, which increases Akt phosphorylation, and consequentially upregulation of eNOS-derived NO production. PMID:27941804

  14. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    SciTech Connect

    Yang, Lei; Wu, Zhong; Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang; Wang, Jianguang; Zhu, Jianguo

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  15. Carbohydrate-binding motif in Chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

    PubMed Central

    Chen, Chun-Chuan; Llado, Victoria; Eurich, Katrin; Tran, Hoa T.; Mizoguchi, Emiko

    2011-01-01

    Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th–339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis. PMID:21546314

  16. Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells.

    PubMed

    Abeyrathna, Prasanna; Kovacs, Laszlo; Han, Weihong; Su, Yunchao

    2016-07-01

    Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.

  17. The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

    PubMed Central

    Rawat, Siddhartha

    2014-01-01

    ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

  18. Annexin 2 Regulates Endothelial Morphogenesis by Controlling AKT Activation and Junctional Integrity*

    PubMed Central

    Su, Shih-Chi; Maxwell, Steve A.; Bayless, Kayla J.

    2010-01-01

    Sprouting angiogenesis is a multistep process that involves endothelial cell activation, basement membrane degradation, proliferation, lumen formation, and stabilization. In this study, we identified annexin 2 as a regulator of endothelial morphogenesis using a three-dimensional in vitro model where sprouting angiogenesis was driven by sphingosine 1-phosphate and angiogenic growth factors. We observed that sphingosine 1-phosphate triggered annexin 2 translocation from the cytosol to the plasma membrane and its association with vascular endothelial (VE)-cadherin. In addition, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions with EGTA, antibodies to the extracellular domain of VE-cadherin, or gene silencing all resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced endothelial sprouting responses observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt. PMID:20947498

  19. Ribonuclease 5 facilitates corneal endothelial wound healing via activation of PI3-kinase/Akt pathway

    PubMed Central

    Kim, Kyoung Woo; Park, Soo Hyun; Lee, Soo Jin; Kim, Jae Chan

    2016-01-01

    To maintain corneal transparency, corneal endothelial cells (CECs) exert a pump function against aqueous inflow. However, human CECs are arrested in the G1-phase and non-proliferative in vivo. Thus, treatment of corneal endothelial decompensation is limited to corneal transplantation, and grafts are vulnerable to immune rejection. Here, we show that ribonuclease (RNase) 5 is more highly expressed in normal human CECs compared to decompensated tissues. Furthermore, RNase 5 up-regulated survival of CECs and accelerated corneal endothelial wound healing in an in vitro wound of human CECs and an in vivo cryo-damaged rabbit model. RNase 5 treatment rapidly induced accumulation of cytoplasmic RNase 5 into the nucleus, and activated PI3-kinase/Akt pathway in human CECs. Moreover, inhibition of nuclear translocation of RNase 5 using neomycin reversed RNase 5-induced Akt activation. As a potential strategy for proliferation enhancement, RNase 5 increased the population of 5-bromo-2′-deoxyuridine (BrdU)-incorporated proliferating CECs with concomitant PI3-kinase/Akt activation, especially in CECs deprived of contact-inhibition. Specifically, RNase 5 suppressed p27 and up-regulated cyclin D1, D3, and E by activating PI3-kinase/Akt in CECs to initiate cell cycle progression. Together, our data indicate that RNase 5 facilitates corneal endothelial wound healing, and identify RNase 5 as a novel target for therapeutic exploitation. PMID:27526633

  20. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  1. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  2. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  3. Polydatin improves glucose and lipid metabolism in experimental diabetes through activating the Akt signaling pathway.

    PubMed

    Hao, Jie; Chen, Cheng; Huang, Kaipeng; Huang, Junying; Li, Jie; Liu, Peiqing; Huang, Heqing

    2014-12-15

    Recently, the effect of polydatin on lipid regulation has gained considerable attention. And previous study has demonstrated that polydatin has hypoglycemic effect on experimental diabetic rats. Repressed Akt pathway contributes to glucose and lipid disorders in diabetes. Thus, whether polydatin regulates glucose and lipid metabolism in experimental diabetic models through the Akt pathway arouses interest. The purpose was to explore the regulatory mechanism of polydain on glucose and lipid through Akt pathway. We used a diabetic rat model induced by high-fat and -sugar diet with low-dose of streptozocin and an insulin resistant HepG2 cell model induced by palmitic acid to clarify the role of polydatin on glucose and lipid metabolism. Here, we found that polydatin significantly attenuated fasting blood–glucose, glycosylated hemoglobin, glycosylated serum protein, total cholesterol, triglyceride, and low-density lipoprotein cholesterol in diabetic rats. Furthermore, polydatin significantly increased glucose uptake and consumption and decreased lipid accumulation in insulin resistant HepG2 cells. Polydatin markedly increased serum insulin levels in diabetic rats, and obviously activated the Akt signaling pathway in diabetic rat livers and insulin resistant HepG2 cells. Polydatin markedly increased phosphorylated GSK-3β, decreased the protein levels of G6Pase and SREBP-1c, and increased protein levels of GCK, LDLR, and phosphorylated IRS in livers and HepG2 cells. Overall, the results indicate that polydatin regulates glucose and lipid metabolism in experimental diabetic models, the underlying mechanism is probably associated with regulating the Akt pathway. The effect of polydatin on increased Akt phosphorylation is independent of prompting insulin secretion, but dependent of increasing IRS phosphorylation.

  4. Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

    SciTech Connect

    Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao; Tan, Guo-Wei; Wang, Zhan-Xiang

    2016-03-18

    Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression of Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.

  5. SYNERGISTIC PROAPOPTOTIC ACTIVITY OF RECOMBINANT TRAIL PLUS THE AKT INHIBITOR PERIFOSINE IN ACUTE MYELOGENOUS LEUKEMIA CELLS

    PubMed Central

    Tazzari, Pier Luigi; Tabellini, Giovanna; Ricci, Francesca; Papa, Veronica; Bortul, Roberta; Chiarini, Francesca; Evangelisti, Camilla; Martinelli, Giovanni; Bontadini, Andrea; Cocco, Lucio; McCubrey, James A.; Martelli, Alberto M.

    2008-01-01

    To potentiate the response of acute myelogenous leukemia (AML) cells to TNF-Related Apoptosis-Inducing Ligand (TRAIL) cytotoxicity, we have examined the efficacy of a combination with perifosine, a novel phosphatidylinositol 3-kinase (PI3K)/Akt signaling inhibitor. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease the cellular FLICE-Inhibitory Protein (cFLIP) in human lung cancer cell lines. Perifosine and TRAIL both induced cell death by apoptosis in the THP-1 AML cell line, which is characterized by constitutive PI3K/Akt activation, but lacks functional p53. Perifosine, at concentrations below IC50, dephosphorylated Akt and increased TRAIL-R2 levels, as demonstrated by western blot, RT-PCR, and flow cytometric analysis. Perifosine also decreased the long isoform of cFLIP (cFLIP-L) and the X-linked Inhibitor of Apoptosis Protein (XIAP) expression. Perifosine and TRAIL synergized to activate caspase-8 and induce apoptosis, which was blocked by a caspase- 8 selective inhibitor. Upregulation of TRAIL-R2 expression was dependent on a protein kinase Cα/c-Jun-NH2-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine synergized with TRAIL also in primary AML cells displaying constitutive activation of the Akt pathway, by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 upregulation, cFLIP-L and XIAP downregulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients. In contrast, CD34+ cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination perifosine and TRAIL might offer a novel therapeutic strategy for AML. PMID:19010914

  6. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

    SciTech Connect

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.

  7. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

    PubMed

    Markova, B; Albers, C; Breitenbuecher, F; Melo, J V; Brümmendorf, T H; Heidel, F; Lipka, D; Duyster, J; Huber, C; Fischer, T

    2010-02-04

    In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

  8. Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

    PubMed

    Hales, Eric C; Orr, Steven M; Larson Gedman, Amanda; Taub, Jeffrey W; Matherly, Larry H

    2013-08-02

    Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

  9. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals

    PubMed Central

    Kureishi, Yasuko; Luo, Zhengyu; Shiojima, Ichiro; Bialik, Ann; Fulton, David; Lefer, David J.; Sessa, William C.; Walsh, Kenneth

    2010-01-01

    Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth. PMID:10973320

  10. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1.

    PubMed

    Wittmann, Torsten; Bokoch, Gary M; Waterman-Storer, Clare M

    2004-02-13

    In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.

  11. p62 modulates Akt activity via association with PKC{zeta} in neuronal survival and differentiation

    SciTech Connect

    Joung, Insil . E-mail: ijoung@hanseo.ac.kr; Kim, Hak Jae; Kwon, Yunhee Kim . E-mail: kimyh@khu.ac.kr

    2005-08-26

    p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKC{zeta} by association of p62 and PKC{zeta}, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation.

  12. Src kinase integrates PI3K/Akt and MAPK/ERK1/2 pathways in T3-induced Na-K-ATPase activity in adult rat alveolar cells.

    PubMed

    Lei, Jianxun; Ingbar, David H

    2011-11-01

    We previously reported that the 3,5,3'-triiodo-L-thyronine (T3)-induced increase of Na-K-ATPase activity in rat alveolar epithelial cells (AECs) required activation of Src kinase, PI3K, and MAPK/ERK1/2. In the present study, we assessed the role of Akt in Na-K-ATPase activity and the interaction between the PI3K and MAPK in response to T3 by using MP48 cells, inhibitors, and constitutively active mutants in the MP48 (alveolar type II-like) cell line. The Akt inhibitor VIII blocked T3-induced increases in Na-K-ATPase activity and amount of plasma membrane Na-K-ATPase protein. The Akt inhibitor VIII also abolished the increase in Na-K-ATPase activity induced by constitutively active mutants of either Src kinase or PI3K. Moreover, constitutively active mutants of Akt increased Na-K-ATPase activity in the absence of T3. Thus activation of Akt was required for T3-induced Na-K-ATPase activity in AECs and is sufficient in the absence of T3. Inhibitors of Src kinase (PP1), PI3K (wortmannin), and ERK1/2 (U0126) all blocked the T3-induced Na-K-ATPase activity. PP1 blocked the activation of PI3K and also ERK1/2 by T3, whereas U0126 did not prevent T3 activation of Src kinase or PI3K activity. Wortmannin did not significantly alter T3-increased MAPK/ERK1/2 activity, suggesting that T3-activated PI3K/Akt and MAPK/ERK1/2 pathways acted downstream of the Src kinase. Furthermore, in the absence of T3, a constitutively active mutant of Src kinase increased activities of Na-K-ATPase, PI3K, and MAPK/ERK1/2. A constitutively active mutant of PI3K enhanced Na-K-ATPase activity but did not alter the MAPK/ERK1/2 activity significantly. In summary, in adult rat AECs T3-stimulated Src kinase activity can activate both PI3K/Akt and MAPK/ERK1/2, and activation of Akt is necessary for T3-induced Na-K-ATPase activity.

  13. Interleukin-18 directly protects cortical neurons by activating PI3K/AKT/NF-κB/CREB pathways.

    PubMed

    Zhou, Jia; Ping, Feng-feng; Lv, Wen-ting; Feng, Jun-yi; Shang, Jing

    2014-09-01

    Interleukin-18 (IL-18), a member of the IL-1 family of cytokines, was initially identified as an interferon (IFN)-γ-inducing factor. IL-18 is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the effects of IL-18 on cortical neurons have not been explored. The present study was conducted to investigate the influence of IL-18 on rat primary cortical neurons and elucidate the underlying mechanisms. We proved that rrIL-18 increased the brain-derived neurotrophic factor (BDNF) expression in a time-dependent manner. Treatment with rrIL-18 (50 ng/ml) deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by facilitating its phosphorylation, enhanced the expression of Phosphoinositide 3-OH kinase (PI3K) and p-Akt, standing for the activation of the PI3K/Akt pathway. As its pivotal downstream pathways, nuclear factor-kappa B (NF-κB), cAMP-responsive element binding protein (CREB)/Bcl-2 and glycogen synthase kinase-3β (GSK-3β) were examined in further steps. Our data revealed that rrIL-18 stimulated NF-κB activation, improved p-CREB and anti-apoptotic Bcl-2 expression levels. But rrIL-18 had little or no effect on GSK-3β pathway. Besides, rrIL-18 increased levels of BDNF and Bcl-2/Bax ratio and decreased cleaved caspase-3 expression to protect cortical neurons from damage induced by oxygen-glucose deprivation (OGD). These results in vitro showed the protection of IL-18 on cortical neurons. And this direct neuroprotective effect of IL-18 is crippled by PI3K inhibitor wortmannin.

  14. Antibiotic drug tigecycline reduces neuroblastoma cells proliferation by inhibiting Akt activation in vitro and in vivo.

    PubMed

    Zhong, Xiaoxia; Zhao, Erhu; Tang, Chunling; Zhang, Weibo; Tan, Juan; Dong, Zhen; Ding, Han-Fei; Cui, Hongjuan

    2016-06-01

    As the first member of glycylcycline bacteriostatic agents, tigecycline is approved as a novel expanded-spectrum antibiotic, which is clinically available. However, accumulating evidence indicated that tigecycline was provided with the potential application in cancer therapy. In this paper, tigecycline was shown to exert an anti-proliferative effect on neuroblastoma cell lines. Furthermore, it was found that tigecycline induced G1-phase cell cycle arrest instead of apoptosis by means of Akt pathway inhibition. In neuroblastoma cell lines, the Akt activator insulin-like growth factor-1 (hereafter referred to as IGF-1) reversed tigecycline-induced cell cycle arrest. Besides, tigecycline inhibited colony formation and suppressed neuroblastoma cells xenograft formation and growth. After tigecycline treatment in vivo, the Akt pathway inhibition was confirmed as well. Collectively, our data provided strong evidences that tigecycline inhibited neuroblastoma cells growth and proliferation through the Akt pathway inhibition in vitro and in vivo. In addition, these results were supported by previous studies concerning the application of tigecycline in human tumors treatment, suggesting that tigecycline might act as a potential candidate agent for neuroblastoma treatment.

  15. Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

    PubMed

    Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

    2014-02-01

    Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

  16. Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy.

    PubMed

    Moc, Courtney; Taylor, Amy E; Chesini, Gino P; Zambrano, Cristina M; Barlow, Melissa S; Zhang, Xiaoxue; Gustafsson, Åsa B; Purcell, Nicole H

    2015-02-01

    To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. To investigate the in vivo requirement for 'physiological' control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  17. SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling.

    PubMed

    Gong, Yi-Qing; Huang, Wei; Li, Ke-Ran; Liu, Yuan-Yuan; Cao, Guo-Fan; Cao, Cong; Jiang, Qin

    2016-09-13

    Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis.

  18. SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

    PubMed Central

    Cao, Guo-fan; Cao, Cong; Jiang, Qin

    2016-01-01

    Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis. PMID:27517753

  19. Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways.

    PubMed

    Tong, Wei; Zhang, Jing; Lodish, Harvey F

    2005-06-15

    Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk(-/-) mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis.

  20. Activation of Akt/FKHR in the medulla oblongata contributes to spontaneous respiratory recovery after incomplete spinal cord injury in adult rats.

    PubMed

    Felix, M S; Bauer, S; Darlot, F; Muscatelli, F; Kastner, A; Gauthier, P; Matarazzo, V

    2014-09-01

    After incomplete spinal cord injury (SCI), patients and animals may exhibit some spontaneous functional recovery which can be partly attributed to remodeling of injured neural circuitry. This post-lesion plasticity implies spinal remodeling but increasing evidences suggest that supraspinal structures contribute also to the functional recovery. Here we tested the hypothesis that partial SCI may activate cell-signaling pathway(s) at the supraspinal level and that this molecular response may contribute to spontaneous recovery. With this aim, we used a rat model of partial cervical hemisection which injures the bulbospinal respiratory tract originating from the medulla oblongata of the brainstem but leads to a time-dependent spontaneous functional recovery of the paralyzed hemidiaphragm. We first demonstrate that after SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata of the brainstem, resulting in an inactivation of its pro-apoptotic downstream target, forkhead transcription factor (FKHR/FOXO1A). Retrograde labeling of medullary premotoneurons including respiratory ones which project to phrenic motoneurons reveals an increased FKHR phosphorylation in their cell bodies together with an unchanged cell number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the SCI-induced Akt and FKHR phosphorylations and activates one of its death-promoting downstream targets, Fas ligand. Quantitative EMG analyses of diaphragmatic contractility demonstrate that the inhibition of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery normally observed after partial cervical SCI. Such inhibition does not however affect either baseline contractile frequency or the ventilatory reactivity under acute respiratory challenge. Together, these findings provide novel evidence of supraspinal cellular contribution to the spontaneous respiratory recovery after partial SCI.

  1. IGF-1 protects oligodendrocyte progenitors against TNFalpha-induced damage by activation of PI3K/Akt and interruption of the mitochondrial apoptotic pathway.

    PubMed

    Pang, Yi; Zheng, Baoying; Fan, Lir-Wan; Rhodes, Philip G; Cai, Zhengwei

    2007-08-15

    Proinflammatory cytokine-mediated injury to oligodendrocyte progenitor cells (OPCs) has been proposed as a cause of periventricular leukomalacia (PVL), the most common brain injury found in preterm infants. Preventing death of OPCs is a potential strategy to prevent or treat PVL. In the current study, we utilized an in vitro cell culture system to investigate the effect of insulin-like growth factor-1 (IGF-1) on tumor necrosis factor-alpha (TNFalpha)-induced OPC injury and the possible mechanisms involved. OPCs were isolated from neonatal rat optic nerves and cultured in chemically defined medium (CDM) supplemented with platelet-derived growth factor and basic fibroblast growth factor. Exposure to TNFalpha resulted in death of OPCs. IGF-1 protected OPCs from TNFalpha cytotoxicity in a dose-dependent manner as measured by the XTT and TUNEL assays. IGF-1 activates both the PI3K/Akt and the extracellular signal-regulated kinase (ERK) pathway. However, IGF-1-enhanced cell survival signals were mediated by the PI3K/Akt, but not by the ERK pathway, as evidenced by the observation that IGF-1-enhanced cell survival was partially abrogated by Akti, the Akt inhibitor, or wortmannin, the PI3K inhibitor, but not by PD98,059, the MAPK kinase/ERK kinase inhibitor. The downstream events of IGF-1-triggered survival signals included phosphorylation of BAD, blockade of TNFalpha-induced translocation of Bax from the cytosol to the mitochondrial membrane, and suppression of caspase-9 and caspase-3 activation. These observations indicate that the protection of OPCs by IGF-1 is mediated, at least partially, by interruption of the mitochondrial apoptotic pathway via activation of PI3K/Akt.

  2. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    PubMed

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-04

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  3. Cytokine Stimulation Promotes Glucose Uptake via Phosphatidylinositol-3 Kinase/Akt Regulation of Glut1 Activity and Trafficking

    PubMed Central

    Wieman, Heather L.; Wofford, Jessica A.

    2007-01-01

    Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization. PMID:17301289

  4. Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

    PubMed

    Zheng, Jianjian; Wu, Cunzao; Xu, Ziqiang; Xia, Peng; Dong, Peihong; Chen, Bicheng; Yu, Fujun

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-β1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

  5. Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

    PubMed Central

    Cuadrado, I; Fernández-Velasco, M; Boscá, L; de las Heras, B

    2011-01-01

    Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis. PMID:22071634

  6. Downstream Effect of Ramping Neuronal Activity through Synapses with Short-Term Plasticity.

    PubMed

    Wei, Wei; Wang, Xiao-Jing

    2016-04-01

    Ramping neuronal activity refers to spiking activity with a rate that increases quasi-linearly over time. It has been observed in multiple cortical areas and is correlated with evidence accumulation processes or timing. In this work, we investigated the downstream effect of ramping neuronal activity through synapses that display short-term facilitation (STF) or depression (STD). We obtained an analytical result for a synapse driven by deterministic linear ramping input that exhibits pure STF or STD and numerically investigated the general case when a synapse displays both STF and STD. We show that the analytical deterministic solution gives an accurate description of the averaging synaptic activation of many inputs converging onto a postsynaptic neuron, even when fluctuations in the ramping input are strong. Activation of a synapse with STF shows an initial cubical increase with time, followed by a linear ramping similar to a synapse without STF. Activation of a synapse with STD grows in time to a maximum before falling and reaching a plateau, and this steady state is independent of the slope of the ramping input. For a synapse displaying both STF and STD, an increase in the depression time constant from a value much smaller than the facilitation time constant τ(F) to a value much larger than τ(F) leads to a transition from facilitation dominance to depression dominance. Therefore, our work provides insights into the impact of ramping neuronal activity on downstream neurons through synapses that display short-term plasticity. In a perceptual decision-making process, ramping activity has been observed in the parietal and prefrontal cortices, with a slope that decreases with task difficulty. Our work predicts that neurons downstream from such a decision circuit could instead display a firing plateau independent of the task difficulty, provided that the synaptic connection is endowed with short-term depression.

  7. Repetitive exposure to low-dose X-irradiation attenuates testicular apoptosis in type 2 diabetic rats, likely via Akt-mediated Nrf2 activation

    PubMed Central

    Zhao, Yuguang; Kong, Chuipeng; Chen, Xiao; Wang, Zhenyu; Wan, Zhiqiang; Jia, Lin; Liu, Qiuju; Wang, Yuehui; Li, Wei; Cui, Jiuwei; Han, Fujun; Cai, Lu

    2017-01-01

    To determine whether repetitive exposure to low-dose radiation (LDR) attenuates type 2 diabetes (T2DM)-induced testicular apoptotic cell death in a T2DM rat model, we examined the effects of LDR exposure on diabetic and age-matched control rats. We found that testicular apoptosis and oxidative stress levels were significantly higher in T2DM rats than in control rats. In addition, glucose metabolism-related Akt and GSK-3β function was downregulated and Akt negative regulators PTP1B and TRB3 were upregulated in the T2DM group. Superoxide dismutase (SOD) activity and catalase content were also found to be decreased in T2DM rats. These effects were partially prevented or reversed by repetitive LDR exposure. Nrf2 and its downstream genes NQO1, SOD, and catalase were significantly upregulated by repetitive exposure to LDR, suggesting that the reduction of T2DM-induced testicular apoptosis due to repetitive LDR exposure likely involves enhancement of testicular Akt-mediated glucose metabolism and anti-oxidative defense mechanisms. PMID:26704079

  8. Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase

    PubMed Central

    Ahn, Jee-Yin; Liu, Xia; Liu, Zhixue; Pereira, Lorena; Cheng, Dongmei; Peng, Junmin; Wade, Paul A; Hamburger, Anne W; Ye, Keqiang

    2006-01-01

    Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1. PMID:16642037

  9. Activation of spinal chemokine receptor CXCR3 mediates bone cancer pain through an Akt-ERK crosstalk pathway in rats.

    PubMed

    Guan, Xue-Hai; Fu, Qiao-Chu; Shi, Dai; Bu, Hui-Lian; Song, Zhen-Peng; Xiong, Bing-Rui; Shu, Bin; Xiang, Hong-Bing; Xu, Bing; Manyande, Anne; Cao, Fei; Tian, Yu-Ke

    2015-01-01

    Previously, we showed that activation of the spinal CXCL9, 10/CXCR3 pathway mediated bone cancer pain (BCP) in rats. However, the cellular mechanism involved is poorly understood. Here, we found that the activated CXCR3 was co-localized with either neurons, microglia, and astrocytes in the spinal cord, or non-peptidergic-, peptidergic-, and A-type neurons in the dorsal root ganglion. The inoculation of Walker-256 mammary gland carcinoma cells into the rat's tibia induced a time-dependent phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2) in the spinal cord, and CXCR3 was necessary for the phosphorylation of Akt and ERK 1/2. Meanwhile, CXCR3 was co-localized with either pAkt or pERK1/2. Blockage of either Akt or ERK1/2 prevented or reversed the mechanical allodynia in BCP rats. Furthermore, there was cross-activation between PI3K/Akt and Raf/MEK/ERK pathway under the BCP condition. Our results demonstrated that the activation of spinal chemokine receptor CXCR3 mediated BCP through Akt and ERK 1/2 kinase, and also indicated a crosstalk between PI3K/Akt and Raf/MEK/ERK signaling pathways under the BCP condition.

  10. AKT activation promotes PTEN hamartoma tumor syndrome–associated cataract development

    PubMed Central

    Sellitto, Caterina; Li, Leping; Gao, Junyuan; Robinson, Michael L.; Lin, Richard Z.; Mathias, Richard T.; White, Thomas W.

    2013-01-01

    Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients. PMID:24270425

  11. Resveratrol Inhibits Cardiac Hypertrophy via AMP-activated Protein Kinase and Akt*

    PubMed Central

    Chan, Anita Y. M.; Dolinsky, Vernon W.; Soltys, Carrie-Lynn M.; Viollet, Benoit; Baksh, Shairaz; Light, Peter E.; Dyck, Jason R. B.

    2008-01-01

    Whereas studies involving animal models of cardiovascular disease demonstrated that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved have not been elucidated. Because studies in cells other than cardiomyocytes revealed that AMP-activated protein kinase (AMPK) and Akt are affected by resveratrol, we hypothesized that resveratrol prevents cardiac myocyte hypertrophy via these two kinase systems. Herein, we demonstrate that resveratrol reduces phenylephrine-induced protein synthesis and cell growth in rat cardiac myocytes via alterations of intracellular pathways involved in controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2). Additionally, we demonstrate that resveratrol negatively regulates the calcineurin-nuclear factor of activated T cells pathway thus modifying a critical component of the transcriptional mechanism involved in pathological cardiac hypertrophy. Our data also indicate that these effects of resveratrol are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is dependent on the presence of the AMPK kinase, LKB1. Taken together, our data suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene transcription. PMID:18562309

  12. Resveratrol inhibits cardiac hypertrophy via AMP-activated protein kinase and Akt.

    PubMed

    Chan, Anita Y M; Dolinsky, Vernon W; Soltys, Carrie-Lynn M; Viollet, Benoit; Baksh, Shairaz; Light, Peter E; Dyck, Jason R B

    2008-08-29

    Whereas studies involving animal models of cardiovascular disease demonstrated that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved have not been elucidated. Because studies in cells other than cardiomyocytes revealed that AMP-activated protein kinase (AMPK) and Akt are affected by resveratrol, we hypothesized that resveratrol prevents cardiac myocyte hypertrophy via these two kinase systems. Herein, we demonstrate that resveratrol reduces phenylephrine-induced protein synthesis and cell growth in rat cardiac myocytes via alterations of intracellular pathways involved in controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2). Additionally, we demonstrate that resveratrol negatively regulates the calcineurin-nuclear factor of activated T cells pathway thus modifying a critical component of the transcriptional mechanism involved in pathological cardiac hypertrophy. Our data also indicate that these effects of resveratrol are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is dependent on the presence of the AMPK kinase, LKB1. Taken together, our data suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene transcription.

  13. EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts.

    PubMed

    Fan, Jian-Bo; Liu, Wei; Zhu, Xin-Hui; Yuan, Kun; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

    2015-01-01

    Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.

  14. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling.

    PubMed

    Yang, Lei; Wu, Zhong; Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang; Wang, Jianguang; Zhu, Jianguo

    2014-12-12

    Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H₂O₂). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H₂O₂. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H₂O₂ was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H₂O₂ activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H₂O₂-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  15. A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development.

    PubMed

    Ciarlo, Christie; Kaufman, Charles K; Kinikoglu, Beste; Michael, Jonathan; Yang, Song; D Amato, Christopher; Blokzijl-Franke, Sasja; den Hertog, Jeroen; Schlaeger, Thorsten M; Zhou, Yi; Liao, Eric; Zon, Leonard I

    2017-08-23

    The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

  16. A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development

    PubMed Central

    Ciarlo, Christie; Kaufman, Charles K; Kinikoglu, Beste; Michael, Jonathan; Yang, Song; D′Amato, Christopher; Blokzijl-Franke, Sasja; den Hertog, Jeroen; Schlaeger, Thorsten M; Zhou, Yi; Liao, Eric

    2017-01-01

    The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation. PMID:28832322

  17. Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells.

    PubMed

    Moore, Jessy; Megaly, Mark; MacNeil, Adam J; Klentrou, Panagiota; Tsiani, Evangelia

    2016-10-01

    Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival. In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events. Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP. Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels. In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

  18. Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats

    PubMed Central

    Wang, Haiyan; Arias, Edward B.

    2016-01-01

    Skeletal muscle insulin resistance is associated with many common age-related diseases, but moderate calorie restriction (CR) can substantially elevate glucose uptake by insulin-stimulated skeletal muscle from both young and old rats. The current study evaluated the isolated epitrochlearis muscle from ∼24.5-mo-old rats that were either fed ad libitum (AL) or subjected to CR (consuming ∼65% of ad libitum, AL, intake beginning at ∼22.5 mo old). Some muscles were also incubated with MK-2206, a potent and selective Akt inhibitor. The most important results were that in isolated muscles, CR vs. AL resulted in 1) greater insulin-stimulated glucose uptake 2) that was accompanied by significantly increased insulin-mediated activation of Akt2, as indicated by greater phosphorylation on both Thr309 and Ser474 along with greater Akt2 activity, 3) concomitant with enhanced phosphorylation of several Akt substrates, including an Akt substrate of 160 kDa on Thr642 and Ser588, filamin C on Ser2213 and proline-rich Akt substrate of 40 kDa on Thr246, but not TBC1D1 on Thr596; and 4) each of the CR effects was eliminated by MK-2206. These data provide compelling new evidence linking greater Akt2 activation to the CR-induced elevation of insulin-stimulated glucose uptake by muscle from old animals. PMID:26739650

  19. Effects of AFP-activated PI3K/Akt signaling pathway on cell proliferation of liver cancer.

    PubMed

    Zheng, Lu; Gong, Wei; Liang, Ping; Huang, XiaoBing; You, Nan; Han, Ke Qiang; Li, Yu Ming; Li, Jing

    2014-05-01

    This study aims to investigate effects of alpha-fetoprotein (AFP)-activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway on hepatocellular carcinoma cell proliferation. Active cirrhosis patients after hepatitis B infection (n = 20) and viral hepatitis patients with hepatocellular carcinoma (HCC) (n = 20) were selected as the subjects of the present study. Another 20 healthy subjects were selected as the control group. The serum AFP expression and liver tissue PI3K and Akt gene mRNA expression were detected. The hepatoma cell model HepG2 which had a stable expression of AFP gene was used. Real-time quantitative PCR and Western blot and other methods were used to analyze the intracellular PI3K and Akt protein levels. Compared with control group and cirrhosis group, the serum AFP levels in HCC group significantly increased, and the tissue PI3K and Akt mRNA expression also significantly increased. HepG2 cells were intervened using AFP, in which the PIK and Akt protein expression significantly increased. After intervention by use of AFP monoclonal antibodies or LY294002 inhibitor, the PIK and Akt protein expression in HepG2 cell was significantly decreased (P < 0.05). AFP can promote the proliferation of hepatoma cells via activation of PI3K/Akt signaling pathway.

  20. Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression.

    PubMed

    Liu, Danyang; Zhang, Yonglong; Wei, Yingze; Liu, Guoyuan; Liu, Yufeng; Gao, Qiongmei; Zou, Liping; Zeng, Wenjiao; Zhang, Nong

    2016-10-04

    Nuclear factor erythroid-2-related factor 2 (Nrf2), a master transcription factor in the antioxidant response, has been found to be ubiquitously expressed in various cancer cells and in the regulation tumor proliferation, invasion, and chemoresistance activities. The regulatory roles of Nrf2 in controlling Hepatocellular carcinoma (HCC) progression remain unclear. In this study, we demonstrated that Nrf2 was significantly elevated in HCC cells and tissues and was correlated with poor prognosis of HCCs. Consistently, Nrf2 significantly promoted HCC cell growth both in vitro and in vivo. Further investigation suggested a novel association of Nrf2 with Platelet-Derived Growth Factor-A (PDGFA). Nrf2 promoted PDGFA transcription by recruiting specificity protein 1 (Sp1) to its promoter, resulting in increased activation of the AKT/p21 pathway and cell cycle progression of HCC cells. As a feedback loop, PDGFA enhanced Nrf2 expression and activation in an AKT dependent manner. In line with these findings, expression of Nrf2 and PDGFA were positively correlated in HCC tissues. Taken together, this study uncovers a novel mechanism of the Nrf2/PDGFA regulatory loop that is crucial for AKT-dependent HCC progression, and thereby provides potential targets for HCC therapy.

  1. Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression

    PubMed Central

    Wei, Yingze; Liu, Guoyuan; Liu, Yufeng; Gao, Qiongmei; Zou, Liping; Zeng, Wenjiao; Zhang, Nong

    2016-01-01

    Nuclear factor erythroid-2-related factor 2 (Nrf2), a master transcription factor in the antioxidant response, has been found to be ubiquitously expressed in various cancer cells and in the regulation tumor proliferation, invasion, and chemoresistance activities. The regulatory roles of Nrf2 in controlling Hepatocellular carcinoma (HCC) progression remain unclear. In this study, we demonstrated that Nrf2 was significantly elevated in HCC cells and tissues and was correlated with poor prognosis of HCCs. Consistently, Nrf2 significantly promoted HCC cell growth both in vitro and in vivo. Further investigation suggested a novel association of Nrf2 with Platelet-Derived Growth Factor-A (PDGFA). Nrf2 promoted PDGFA transcription by recruiting specificity protein 1 (Sp1) to its promoter, resulting in increased activation of the AKT/p21 pathway and cell cycle progression of HCC cells. As a feedback loop, PDGFA enhanced Nrf2 expression and activation in an AKT dependent manner. In line with these findings, expression of Nrf2 and PDGFA were positively correlated in HCC tissues. Taken together, this study uncovers a novel mechanism of the Nrf2/PDGFA regulatory loop that is crucial for AKT-dependent HCC progression, and thereby provides potential targets for HCC therapy. PMID:27588483

  2. FoxO1-Mediated Activation of Akt Plays a Critical Role in Vascular Homeostasis

    PubMed Central

    Yuan, Lei; Dupuis, Dylan; Beeler, David; Spokes, Katherine C.; Janes, Lauren; Sciuto, Tracey; Kang, Peter M.; Jaminet, Shou-Ching S.; Dvorak, Ann; Grant, Marianne A.; Regan, Erzsébet Ravasz; Aird, William C.

    2016-01-01

    Rationale Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and a number of cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells. Previous studies have shown that FoxO1 knockout in mice results in embryonic lethality at E11 due to impaired vascular development. In contrast, somatic deletion of FoxO1 is associated with hyperproliferation of endothelial cells. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. Objective To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. Methods and Results We show that endothelial cell (EC)-specific disruption of FoxO1 in mice phenocopies the full knockout. While endothelial expression of FoxO1 rescued otherwise FoxO-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and VEGF-induced Akt-mTOR1 signaling. Conclusions Our findings suggest that in mice endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor-responsive Akt/mTORC1 activity within a homeostatic range. PMID:24874427

  3. Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium.

    PubMed

    Liu, Wei; Yin, Tuanfang; Ren, Jihao; Li, Lihua; Xiao, Zian; Chen, Xing; Xie, Dinghua

    2014-02-01

    Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.

  4. Essential role of AKT in tumor cells addicted to FGFR.

    PubMed

    Hu, Yi; Lu, Huiru; Zhang, Jinchao; Chen, Jun; Chai, Zhifang; Zhang, Jingxin

    2014-02-01

    Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors.

  5. Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

    PubMed

    Cui, Yue; Zhang, X Q; Cui, Y; Xin, W J; Jing, J; Liu, X G

    2010-11-24

    Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

  6. Tetramethylpyrazine reduces glucose and insulin-induced activation of hepatic stellate cells by inhibiting insulin receptor-mediated PI3K/AKT and ERK pathways.

    PubMed

    Zhang, Feng; Zhang, Zili; Kong, Desong; Zhang, Xiaoping; Chen, Li; Zhu, Xiaojing; Lu, Yin; Zheng, Shizhong

    2014-01-25

    Hepatic stellate cell (HSC) activation is the central event during liver fibrogenesis. Metabolic syndrome characterized by hyperglycemia and hyperinsulinemia contributes to nonalcoholic steatohepatitis-associated liver fibrosis. This study was to investigate the effects of tetramethylpyrazine (TMP) on HSC activation induced by glucose and insulin (Glu/Ins) and the underlying mechanisms. Results showed that Glu/Ins significantly stimulated proliferation, invasion, adhesion, and extracellular matrix (ECM) production in HSCs. TMP inhibited HSC proliferation, invasion and adhesion, and reduced the expression of marker genes related to HSC activation in Glu/Ins-activated HSCs. Mechanistic evidence revealed that TMP reduced insulin receptor (InsR) expression and blocked the downstream phosphatidylinositol-3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) cascades, which was required for TMP attenuation of HSC activation. Moreover, TMP modulated the genes relevant to ECM homeostasis favoring ECM degradation. It could be concluded that TMP inhibited Glu/Ins-stimulated HSC activation and ECM production by inhibiting InsR-mediated PI3K/AKT and ERK pathways. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway

    PubMed Central

    Zhang, B; Zhang, B; Chen, X; Bae, S; Singh, K; Washington, M K; Datta, P K

    2014-01-01

    Background: Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown. Methods: We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed. Results: Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays. Conclusion: Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU. PMID:24384683

  8. AKT-phosphorylated FOXO1 suppresses ERK activation and chemoresistance by disrupting IQGAP1-MAPK interaction.

    PubMed

    Pan, Chun-Wu; Jin, Xin; Zhao, Yu; Pan, Yunqian; Yang, Jing; Karnes, R Jeffrey; Zhang, Jun; Wang, Liguo; Huang, Haojie

    2017-03-09

    Nuclear FOXO proteins act as tumor suppressors by transcriptionally activating genes involved in apoptosis and cell cycle arrest, and these anticancer functions are inhibited by AKT-induced phosphorylation and cytoplasmic sequestration of FOXOs. We found that, after AKT-mediated phosphorylation at serine 319, FOXO1 binds to IQGAP1, a hub for activation of the MAPK pathway, and impedes IQGAP1-dependent phosphorylation of ERK1/2 (pERK1/2). Conversely, decreased FOXO1 expression increases pERK1/2 in cancer cell lines and correlates with increased pERK1/2 levels in patient specimens and disease progression. Treatment of cancer cells with PI3K inhibitors or taxane causes FOXO1 localization in the nucleus, increased expression of pERK1/2, and drug resistance. These effects are reversed by administering a small FOXO1-derived phospho-mimicking peptide inhibitor in vitro and in mice. Our results show a tumor suppressor role of AKT-phosphorylated FOXO1 in the cytoplasm and suggest that this function of FOXO1 can be harnessed to overcome chemoresistance in cancer.

  9. Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation

    PubMed Central

    Huang, Xionggao; Zhou, Guohong; Wu, Wenyi; Ma, Gaoen; D'Amore, Patricia A.; Mukai, Shizuo; Lei, Hetian

    2017-01-01

    Purpose Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Methods Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. Results In the VEGFR2-sgRNA/SpCas9–transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9–transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. Conclusions The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis. PMID:28241310

  10. PDGF-D/PDGFRβ promotes tongue squamous carcinoma cell (TSCC) progression via activating p38/AKT/ERK/EMT signal pathway.

    PubMed

    Zhang, Hong; Sun, Jia-Dong; Yan, Ling-Jian; Zhao, Xiao-Peng

    2016-09-16

    Platelet-derived growth factor D (PDGF-D) signaling plays significant roles during the development and progression of human malignancies via interacting with the receptor of PDGF-D (PDGFR). Meanwhile, the majority of human tumor metastasis is closely associated with epithelial-mesenchymal transition (EMT). However, the underlying mechanism between PDGF-D/PDGFR signaling and EMT which involved in tumor metastasis remain dismal. This study aimed to investigate the role of PDGF-D signaling during EMT process of tongue squamous cell carcinoma (TSCC). In our study, the expression of PDGF-D and PDGFR were examined in primary TSCC samples and the expression of PDGF-D was also determined in TSCC cell lines. In addition, the correlation between PDGF-D expression and TSCC aggressive histopathological features was analyzed. Our results implied that upregulation of PDGFRβ in UM1 cells induced with exogenous PDGF-D can remarkably promote tumor cells invasiveness; conversely, when using small interfering RNA (siRNA), the invasiveness can be severely prohibited. Furthermore, PDGF-D downstream signal molecules p38, AKT, ERK and EMT biomarkers (E-cadherin, N-cadherin, Vimentin and snail) were measured using Western blot. Our results showed that PDGF-D can induce p38, AKT and ERK phosphorylation; downregulate epithelial markers and upregulate mesenchymal markers. On the contrary, PDGFRβ siRNA significantly prohibited p38, AKT and ERK phosphorylation; inhibited EMT process. Function analysis revealed that PDGFRβ siRNA obviously interfered with UM1 cell migration and invasion, according to transwell and wound healing assay. In conclusion, this study suggested that EMT process can be triggered by the PDGF-D/PDGFRβ axis in TSCC, and then involved in the tumor cell invasion via activation of p38/AKT/ERK/EMT pathway.

  11. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  12. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  13. Synthesis and SAR study of modulators inhibiting tRXRα-dependent AKT activation

    PubMed Central

    Wang, Zhi-Gang; Chen, Liqun; Chen, Jiebo; Zheng, Jian-Feng; Gao, Weiwei; Zeng, Zhiping; Zhou, Hu; Zhang, Xiao-kun; Huang, Pei-Qiang; Su, Ying

    2013-01-01

    RXRα represents an intriguing and unique target for pharmacologic interventions. We recently showed that Sulindac and a designed analog could bind to RXRα and modulate its biological activity, including inhibition of the interaction of an N-terminally truncated RXRα (tRXRα) with the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K). Here we report the synthesis, testing and SAR of a series of novel analogs of Sulindac as potential modulators for inhibiting tRXRα-dependent AKT activation. A new compound 30 was identified to have improved biological activity. PMID:23434637

  14. Valproic acid induces monoamine oxidase A via Akt/forkhead box O1 activation.

    PubMed

    Wu, Jason Boyang; Shih, Jean C

    2011-10-01

    Valproic acid (VPA) has been widely used in clinics for the treatment of multiple neuropsychiatric disorders, such as epilepsy and bipolar disorder. One of the mechanisms by which VPA exerts its effect is through regulating the brain levels of serotonin. However, the molecular basis of this VPA action is not fully understood. Here, we report for the first time that VPA activates monoamine oxidase (MAO) A catalytic activity, mRNA level, and promoter activity. MAO A is a key enzyme that degrades a number of monoamine neurotransmitters, including serotonin. Our results show that VPA increased the phosphorylation of both Akt and Forkhead box O1 (FoxO1), whereas pretreatment of cells with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (a phosphoinositide 3-kinase inhibitor) reduced the VPA activation of MAO A. Overexpression of FoxO1 dramatically repressed both the basal and VPA-induced MAO A catalytic and promoter activities to 30 to 60%. Small interfering RNA knockdown of FoxO1 attenuated the stimulating effect of VPA on MAO A. Moreover, introduction of a constitutively active form of FoxO1 abolished the activation of MAO A by VPA and Akt. These results suggest that FoxO1 is a repressor for MAO A transcription, and its phosphorylation is involved in VPA activation of MAO A. Sequence analysis, electrophoretic mobility shift and chromatin immunoprecipitation assays further showed the presence of a functional FoxO1-binding site in MAO A core promoter. Taken together, these results demonstrate that MAO A is a novel target for VPA via Akt/FoxO1 signaling pathway. This information provides new insights into the pharmacological mechanisms and therapeutic implications of VPA action.

  15. Valproic Acid Induces Monoamine Oxidase A via Akt/Forkhead Box O1 ActivationS⃞

    PubMed Central

    Wu, Jason Boyang

    2011-01-01

    Valproic acid (VPA) has been widely used in clinics for the treatment of multiple neuropsychiatric disorders, such as epilepsy and bipolar disorder. One of the mechanisms by which VPA exerts its effect is through regulating the brain levels of serotonin. However, the molecular basis of this VPA action is not fully understood. Here, we report for the first time that VPA activates monoamine oxidase (MAO) A catalytic activity, mRNA level, and promoter activity. MAO A is a key enzyme that degrades a number of monoamine neurotransmitters, including serotonin. Our results show that VPA increased the phosphorylation of both Akt and Forkhead box O1 (FoxO1), whereas pretreatment of cells with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (a phosphoinositide 3-kinase inhibitor) reduced the VPA activation of MAO A. Overexpression of FoxO1 dramatically repressed both the basal and VPA-induced MAO A catalytic and promoter activities to 30 to 60%. Small interfering RNA knockdown of FoxO1 attenuated the stimulating effect of VPA on MAO A. Moreover, introduction of a constitutively active form of FoxO1 abolished the activation of MAO A by VPA and Akt. These results suggest that FoxO1 is a repressor for MAO A transcription, and its phosphorylation is involved in VPA activation of MAO A. Sequence analysis, electrophoretic mobility shift and chromatin immunoprecipitation assays further showed the presence of a functional FoxO1-binding site in MAO A core promoter. Taken together, these results demonstrate that MAO A is a novel target for VPA via Akt/FoxO1 signaling pathway. This information provides new insights into the pharmacological mechanisms and therapeutic implications of VPA action. PMID:21775495

  16. Negative Immune Regulator TIPE2 Promotes M2 Macrophage Differentiation through the Activation of PI3K-AKT Signaling Pathway

    PubMed Central

    Geng, Wenwen; Chen, Youhai H.; Zhang, Cui

    2017-01-01

    Macrophages play important roles in the regulation of the innate and adaptive immune responses. Classically activated macrophages and alternatively activated macrophages are the two major forms of macrophages and have opposing functionalities. Tumor necrosis factor-α-induced protein 8–2 is expressed primarily by immune cells and negatively regulates type 1 innate and adaptive immune responses to maintain immune tolerance. While previous studies indicate that TIPE2 promotes M2 but inhibits M1 macrophage differentiation, the underlying molecular mechanism by which TIPE2 promotes M2 macrophage differentiation remains unclear. Our current study shows that TIPE2-deficient bone-marrow cells are defective in IL-4 induced M2 macrophage differentiation in vitro. Mechanistic studies revealed that TIPE2 promotes phosphoinositide metabolism and the activation of the down-stream AKT signaling pathway, which in turn leads to the expression of markers specific for M2 macrophages. In addition, our results showed that Tipe2-deficiency does not affect the activation of the JAK-STAT6 signaling pathway that also plays an important role during M2 macrophage differentiation. Taken together, these results indicate that TIPE2 promotes M2 macrophage differentiation through the activation of PI3K-AKT signaling pathway, and may play an important role during the resolution of inflammation, parasite control, as well as tissue repair. PMID:28122045

  17. Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages

    PubMed Central

    Chai, Jianshen; Luo, Li; Hou, Fengyan; Fan, Xia; Yu, Jing; Ma, Wei; Tang, Wangqi; Yang, Xue; Zhu, Junyu; Kang, Wenyuan; Yan, Jun; Liang, Huaping

    2016-01-01

    Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. PMID:27685463

  18. The antioxidant compound tert-butylhydroquinone activates Akt in myocardium, suppresses apoptosis and ameliorates pressure overload-induced cardiac dysfunction.

    PubMed

    Zhang, Yongtao; Fang Liu, Fang; Bi, Xiaolei; Wang, Shuangxi; Wu, Xiao; Jiang, Fan

    2015-08-11

    Tert-butylhydroquinone (TBHQ) is an antioxidant compound which shows multiple cytoprotective actions. We evaluated the effects of TBHQ on pathological cardiac remodeling and dysfunction induced by chronic overload. Pressure overload was created by transverse aortic constriction (TAC) in male C57BL/6 mice. TBHQ was incorporated in the diet and administered for 4 weeks. TBHQ treatment prevented left ventricular dilatation and cardiac dysfunction induced by TAC, and decreased the prevalence of myocardial apoptosis. The beneficial effects of TBHQ were associated with an increase in Akt activation, but not related to activations of Nrf2 or AMP-activated protein kinase. TBHQ-induced Akt activation was accompanied by increased phosphorylation of Bad, glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin (mTOR). Mechanistically, we showed that in cultured H9c2 cells and primary cardiac myocytes, TBHQ stimulated Akt phosphorylation and suppressed oxidant-induced apoptosis; this effect was abolished by wortmannin or an Akt inhibitor. Blockade of the Akt pathway in vivo accelerated cardiac dysfunction, and abrogated the protective effects of TBHQ. TBHQ also reduced the reactive aldehyde production and protein carbonylation in stressed myocardium. We suggest that TBHQ treatment may represent a novel strategy for timely activation of the cytoprotective Akt pathway in stressed myocardium.

  19. The antioxidant compound tert-butylhydroquinone activates Akt in myocardium, suppresses apoptosis and ameliorates pressure overload-induced cardiac dysfunction

    PubMed Central

    Zhang, Yongtao; Fang Liu, Fang; Bi, Xiaolei; Wang, Shuangxi; Wu, Xiao; Jiang, Fan

    2015-01-01

    Tert-butylhydroquinone (TBHQ) is an antioxidant compound which shows multiple cytoprotective actions. We evaluated the effects of TBHQ on pathological cardiac remodeling and dysfunction induced by chronic overload. Pressure overload was created by transverse aortic constriction (TAC) in male C57BL/6 mice. TBHQ was incorporated in the diet and administered for 4 weeks. TBHQ treatment prevented left ventricular dilatation and cardiac dysfunction induced by TAC, and decreased the prevalence of myocardial apoptosis. The beneficial effects of TBHQ were associated with an increase in Akt activation, but not related to activations of Nrf2 or AMP-activated protein kinase. TBHQ-induced Akt activation was accompanied by increased phosphorylation of Bad, glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin (mTOR). Mechanistically, we showed that in cultured H9c2 cells and primary cardiac myocytes, TBHQ stimulated Akt phosphorylation and suppressed oxidant-induced apoptosis; this effect was abolished by wortmannin or an Akt inhibitor. Blockade of the Akt pathway in vivo accelerated cardiac dysfunction, and abrogated the protective effects of TBHQ. TBHQ also reduced the reactive aldehyde production and protein carbonylation in stressed myocardium. We suggest that TBHQ treatment may represent a novel strategy for timely activation of the cytoprotective Akt pathway in stressed myocardium. PMID:26260024

  20. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

    PubMed

    Robertson, Anthony J; Coluccio, Alison; Jensen, Sarah; Rydlizky, Katarina; Coffman, James A

    2013-05-15

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  1. Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells*

    PubMed Central

    Patel, Satyananda; Djerdjouri, Bahia; Raoul-Des-Essarts, Yannick; Dang, Pham My-Chan; El-Benna, Jamel; Périanin, Axel

    2010-01-01

    Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (Km 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67phox and p47phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47phox, p67phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. PMID:20693286

  2. Endoglin haploinsufficiency promotes fibroblast accumulation during wound healing through Akt activation.

    PubMed

    Pericacho, Miguel; Velasco, Soraya; Prieto, Marta; Llano, Elena; López-Novoa, José M; Rodríguez-Barbero, Alicia

    2013-01-01

    Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng(+/-)) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng(+/-) MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng(+/-) mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng(+/-) mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.

  3. Endoglin Haploinsufficiency Promotes Fibroblast Accumulation during Wound Healing through Akt Activation

    PubMed Central

    Pericacho, Miguel; Velasco, Soraya; Prieto, Marta; Llano, Elena; López-Novoa, José M.; Rodríguez-Barbero, Alicia

    2013-01-01

    Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng+/−) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng+/− MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng+/− mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng+/− mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds. PMID:23349951

  4. Restoring Akt1 activity in outgrowth endothelial cells from South Asian men rescues vascular reparative potential.

    PubMed

    Cubbon, Richard M; Yuldasheva, Nadira Y; Viswambharan, Hema; Mercer, Ben N; Baliga, Vivek; Stephen, Sam L; Askham, Jonathan; Sukumar, Piruthivi; Skromna, Anna; Mughal, Romana S; Walker, Andrew M N; Bruns, Alexander; Bailey, Marc A; Galloway, Stacey; Imrie, Helen; Gage, Matthew C; Rakobowchuk, Mark; Li, Jing; Porter, Karen E; Ponnambalam, Sreenivasan; Wheatcroft, Stephen B; Beech, David J; Kearney, Mark T

    2014-10-01

    Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC-mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men. © 2014 AlphaMed Press.

  5. Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis.

    PubMed

    Sury, Matthias D; Vorlet-Fawer, Lorianne; Agarinis, Claudia; Yousefi, Shida; Grandgirard, Denis; Leib, Stephen L; Christen, Stephan

    2011-01-01

    Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

  6. Berberine ameliorates hyperglycemia in alloxan-induced diabetic C57BL/6 mice through activation of Akt signaling pathway.

    PubMed

    Xie, Xi; Li, Wenyuan; Lan, Tian; Liu, Weihua; Peng, Jing; Huang, Kaipeng; Huang, Juan; Shen, Xiaoyan; Liu, Peiqing; Huang, Heqing

    2011-01-01

    Recently, it is implicated that the abnormality of Akt signaling pathway is involved in the diabetic pathology. Previous studies have demonstrated that berberine could decrease blood glucose by elevating liver glycogen synthesis. However, the underlying mechanism is still unclear. In the present study, we investigated the effects of berberine on fasting blood glucose, liver glycogen, Akt, Glycogen synthase kinase-3, glucokinase and insulin receptor substrate (IRS) in alloxan-induced diabetic mice, exploring its possible hypoglycemic mechanism. We found that in alloxan-induced diabetic mice, the high blood glucose was significantly lowered by berberine treatment. Liver glycogen content, the expression and activity of glucokinase and the phosphorylated Akt and IRS were all significantly reduced in diabetic mice whereas berberine blocked these changes. Berberine also depressed the increasing of phosphorylated GSK-3β in diabetic mice. Collectively, Berberine upregulates the activity of Akt possibly via insulin signaling pathway, eventually lowering high blood glucose in alloxan-induced diabetic mice.

  7. Three Novel Downstream Promoter Elements Regulate MHC Class I Promoter Activity in Mammalian Cells

    PubMed Central

    Lee, Namhoon; Iyer, Shankar S.; Mu, Jie; Weissman, Jocelyn D.; Ohali, Anat; Howcroft, T. Kevin; Lewis, Brian A.; Singer, Dinah S.

    2010-01-01

    Background MHC class I transcription is regulated by two distinct types of regulatory pathways: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. Methodology/Principal Findings We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3′ end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5′ end of the core promoter. Conclusions/Significance Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5′ end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription. PMID:21179443

  8. Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

    PubMed

    Cardoso, A P; Pinto, M L; Pinto, A T; Oliveira, M I; Pinto, M T; Gonçalves, R; Relvas, J B; Figueiredo, C; Seruca, R; Mantovani, A; Mareel, M; Barbosa, M A; Oliveira, M J

    2014-04-17

    The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

  9. Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

    PubMed Central

    Hoyal, Carolyn R.; Gutierrez, Abel; Young, Brandon M.; Catz, Sergio D.; Lin, Jun-Hsiang; Tsichlis, Philip N.; Babior, Bernard M.

    2003-01-01

    The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47PHOX occurs during the activation of the enzyme in intact cells. p47PHOX carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47PHOX is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47PHOX whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47PHOX by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47PHOX. These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase. PMID:12704229

  10. Activation of Akt1 accelerates carcinogen-induced tumorigenesis in mammary gland of virgin and post-lactating transgenic mice

    PubMed Central

    2014-01-01

    Background Data from in vivo and in vitro studies suggest that activation of Akt regulates cell survival signaling and plays a key role in tumorigenesis. Hence, transgenic mice were created to explore the oncogenic role of Akt1 in the development of mammary tumors. Methods The transgenic mice were generated by expressing myristoylated-Akt1 (myr-Akt1) under the control of the MMTV-LTR promoter. The carcinogen 7, 12 dimethyl-1,2-benzanthracene (DMBA) was used to induce tumor formation. Results The MMTV driven myr-Akt1 transgene expression was detected primarily in the mammary glands, uterus, and ovaries. The expression level increased significantly in lactating mice, suggesting that the response was hormone dependent. The total Akt expression level in the mammary gland was also higher in the lactating mice. Interestingly, the expression of MMTVmyr-Akt1 in the ovaries of the transgenic mice caused significant increase in circulating estrogen levels, even at the post-lactation stage. Expression of myr-Akt1 in mammary glands alone did not increase the frequency of tumor formation. However, there was an increased susceptibility of forming mammary tumors induced by DMBA in the transgenic mice, especially in mice post-lactation. Within 34 weeks, DMBA induced mammary tumors in 42.9% of transgenic mice post-lactation, but not in wild-type mice post-lactation. The myr-Akt1 mammary tumors induced by DMBA had increased phosphorylated-Akt1 and showed strong expression of estrogen receptor (ERα) and epidermal growth factor receptor (EGFR). In addition, Cyclin D1 was more frequently up-regulated in mammary tumors from transgenic mice compared to tumors from wild-type mice. Overexpression of Cyclin D1, however, was not completely dependent on activated Akt1. Interestingly, mammary tumors that had metastasized to secondary sites had increased expression of Twist and Slug, but low expression of Cyclin D1. Conclusions In summary, the MMTVmyr-Akt1 transgenic mouse model could be

  11. Activation of Akt1 accelerates carcinogen-induced tumorigenesis in mammary gland of virgin and post-lactating transgenic mice.

    PubMed

    Wu, Yanyuan; Kim, Juri; Elshimali, Yayha; Sarkissyan, Marianna; Vadgama, Jaydutt V

    2014-04-17

    Data from in vivo and in vitro studies suggest that activation of Akt regulates cell survival signaling and plays a key role in tumorigenesis. Hence, transgenic mice were created to explore the oncogenic role of Akt1 in the development of mammary tumors. The transgenic mice were generated by expressing myristoylated-Akt1 (myr-Akt1) under the control of the MMTV-LTR promoter. The carcinogen 7, 12 dimethyl-1,2-benzanthracene (DMBA) was used to induce tumor formation. The MMTV driven myr-Akt1 transgene expression was detected primarily in the mammary glands, uterus, and ovaries. The expression level increased significantly in lactating mice, suggesting that the response was hormone dependent. The total Akt expression level in the mammary gland was also higher in the lactating mice. Interestingly, the expression of MMTVmyr-Akt1 in the ovaries of the transgenic mice caused significant increase in circulating estrogen levels, even at the post-lactation stage. Expression of myr-Akt1 in mammary glands alone did not increase the frequency of tumor formation. However, there was an increased susceptibility of forming mammary tumors induced by DMBA in the transgenic mice, especially in mice post-lactation. Within 34 weeks, DMBA induced mammary tumors in 42.9% of transgenic mice post-lactation, but not in wild-type mice post-lactation. The myr-Akt1 mammary tumors induced by DMBA had increased phosphorylated-Akt1 and showed strong expression of estrogen receptor (ERα) and epidermal growth factor receptor (EGFR). In addition, Cyclin D1 was more frequently up-regulated in mammary tumors from transgenic mice compared to tumors from wild-type mice. Overexpression of Cyclin D1, however, was not completely dependent on activated Akt1. Interestingly, mammary tumors that had metastasized to secondary sites had increased expression of Twist and Slug, but low expression of Cyclin D1. In summary, the MMTVmyr-Akt1 transgenic mouse model could be useful to study mechanisms of ER

  12. Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

    PubMed

    Mimura, Naoya; Hideshima, Teru; Shimomura, Toshiyasu; Suzuki, Rikio; Ohguchi, Hiroto; Rizq, Ola; Kikuchi, Shohei; Yoshida, Yasuhiro; Cottini, Francesca; Jakubikova, Jana; Cirstea, Diana; Gorgun, Gullu; Minami, Jiro; Tai, Yu-Tzu; Richardson, Paul G; Utsugi, Teruhiro; Iwama, Atsushi; Anderson, Kenneth C

    2014-08-15

    The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. ©2014 American Association for Cancer Research.

  13. Testosterone and Voluntary Exercise, Alone or Together Increase Cardiac Activation of AKT and ERK1/2 in Diabetic Rats

    PubMed Central

    Chodari, Leila; Mohammadi, Mustafa; Mohaddes, Gisou; Alipour, Mohammad Reza; Ghorbanzade, Vajiheh; Dariushnejad, Hassan; Mohammadi, Shima

    2016-01-01

    Background Impaired angiogenesis in cardiac tissue is a major complication of diabetes. Protein kinase B (AKT) and extracellular signal regulated kinase (ERK) signaling pathways play important role during capillary-like network formation in angiogenesis process. Objectives To determine the effects of testosterone and voluntary exercise on levels of vascularity, phosphorylated Akt (P- AKT) and phosphorylated ERK (P-ERK) in heart tissue of diabetic and castrated diabetic rats. Methods Type I diabetes was induced by i.p injection of 50 mg/kg of streptozotocin in animals. After 42 days of treatment with testosterone (2mg/kg/day) or voluntary exercise alone or in combination, heart tissue samples were collected and used for histological evaluation and determination of P-AKT and P-ERK levels by ELISA method. Results Our results showed that either testosterone or exercise increased capillarity, P-AKT, and P-ERK levels in the heart of diabetic rats. Treatment of diabetic rats with testosterone and exercise had a synergistic effect on capillarity, P-AKT, and P-ERK levels in heart. Furthermore, in the castrated diabetes group, capillarity, P-AKT, and P-ERK levels significantly decreased in the heart, whereas either testosterone treatment or exercise training reversed these effects. Also, simultaneous treatment of castrated diabetic rats with testosterone and exercise had an additive effect on P-AKT and P-ERK levels. Conclusion Our findings suggest that testosterone and exercise alone or together can increase angiogenesis in the heart of diabetic and castrated diabetic rats. The proangiogenesis effects of testosterone and exercise are associated with the enhanced activation of AKT and ERK1/2 in heart tissue.

  14. Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux

    PubMed Central

    He, Yuan; Zeng, Melody Y.; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome

  15. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

    PubMed

    He, Yuan; Zeng, Melody Y; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-02-18

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to

  16. Sirt1 Inhibits Akt2-Mediated Porcine Adipogenesis Potentially by Direct Protein-Protein Interaction

    PubMed Central

    Pang, Weijun; Wang, Yu; Wei, Ning; Xu, Ruxiang; Xiong, Yan; Wang, Ping; Shen, Qingwu; Yang, Gongshe

    2013-01-01

    Compared with the rodent, the domestic pig is a much better animal model for studying adipogenesis and obesity-related diseases. Currently, the role of Akt2 and Sirt1 in porcine adipogenesis remains elusive. In this study, we defined the effect of Akt2 and Sirt1 on porcine preadipocyte lipogenesis and the regulatory mechanism. First, we found that Akt2 was widely expressed in porcine various tissues and at high level in adipose tissue. Further analysis showed that the expression level of Akt2 was much higher in adipose tissue and adipocytes of the Bamei pig breed (a Chinese indigenous fatty pig) than in that of the Large White pig breed (a Lean type pig), whereas the level of Sirt1 expression was opposite. The expression levels of Sirt1 and Akt2 gradually increased during adipogenic differentiation. Adipogenesis was robustly inhibited in Akt2 deficient fat cells, whereas it was promoted in Sirt1 deficient cells using the lentiviral–mediated shRNA approach. Interestingly, adipogenesis returned to normal in Akt2 and Sirt1 dual–deficient cells, showing that the pro- and anti–adipogenic effects were balanced. Sirt1 inhibited transcriptional activity of Akt2 in a dose-dependent way. Interaction of endogenous Akt2 and Sirt1 was gradually enhanced before day 6 of differentiation, and then attenuated. Akt2 and Sirt1 also interacted with C/EBPα in adipocytes. Moreover, knockdown of Akt2 or/and Sirt1 affected pro–lipogenesis of insulin–stimulated by PI3K/Akt pathway. We further found that Sirt1 respectively interacted with PI3K and GSK3β which were key upstream and downstream components of PI3K/Akt pathway. Based on the above findings, we concluded that the crosstalk between C/EBPα and PI3K/Akt signaling pathways is implicated in Akt2 and Sirt1 regulation of adipogenesis. PMID:23951196

  17. Withaferin A inhibits matrix metalloproteinase-9 activity by suppressing the Akt signaling pathway.

    PubMed

    Lee, Dae Hyung; Lim, In-Hye; Sung, Eon-Gi; Kim, Joo-Young; Song, In-Hwan; Park, Yoon Ki; Lee, Tae-Jin

    2013-08-01

    Withaferin A (Wit A), a steroidal lactone isolated from Withania somnifera, exhibits anti-inflammatory, immuno-modulatory and anti-angiogenic properties and antitumor activities. In the present study, we investigated the effects of Wit A on protease-mediated invasiveness of the human metastatic cancer cell lines Caski and SK-Hep1. We found that treatment with Wit A resulted in marked inhibition of the TGF‑β‑induced increase in expression and activity of matrix metalloproteinase (MMP)‑9 in Caski cell line. These effects of Wit A were dose-dependent and showed a correlation with suppression of MMP‑9 mRNA expression levels. Treatment with Wit A resulted in an ~1.6-fold induction of MMP-9 promoter activity, which was also suppressed by treatment with Wit A in Caski cells. We found that treatment with Wit A resulted in inhibition of TGF‑β‑induced phosphorylation of Akt, which was involved in the downregulation of expression of MMP-9 at the protein level. Introduction with constitutively active (CA)‑Akt resulted in a partial increase in the secretion of TGF-β-induced MMP-9 blocked by treatment with Wit A in Caski cells. According to these results, Wit A may inhibit the invasive and migratory abilities of Caski cells through a reduction in MMP-9 expression through suppression of the pAkt signaling pathway. These findings indicate that use of Wit A may be an effective strategy for control of metastasis and invasiveness of tumors.

  18. Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

    PubMed

    Lee, Tae Hoon; Jung, Hana; Park, Keun Hyung; Bang, Myun Ho; Baek, Nam-In; Kim, Jiyoung

    2014-10-01

    Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

  19. A Hot-spot of In-frame Duplications Activates the Oncoprotein AKT1 in Juvenile Granulosa Cell Tumors

    PubMed Central

    Bessière, Laurianne; Todeschini, Anne-Laure; Auguste, Aurélie; Sarnacki, Sabine; Flatters, Delphine; Legois, Bérangère; Sultan, Charles; Kalfa, Nicolas; Galmiche, Louise; Veitia, Reiner A.

    2015-01-01

    Background Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of Gαs, which transduces mitogenic signals, in 30% of the analyzed JGCTs. Methods We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K–AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays. Findings We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays. Interpretation Our study incriminates somatic mutations of AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols. PMID:26137586

  20. Association of HOTAIR expression with PI3K/Akt pathway activation in adenocarcinoma of esophagogastric junction

    PubMed Central

    Hui, Zhang

    2016-01-01

    Abstract Objectives Although the Hox transcript antisense intergenic RNA (HOTAIR), a vital long non-coding RNA, is known to participate in the development and progression of a wide range of carcinomas, there are still no published reports regarding its expression in adenocarcinoma of esophagogastric junction (AEJ). The aims of this study were to investigate the expression of HOTAIR, and to analyze the association of its expression with PI3K/Akt pathway activation in clinical AEJ patients. Methods Nine normal epithelial tissues and 41 samples of AEJ were studied comparably. The expression of HOTAIR was detected by real-time PCR according to the different tumor grades in these AEJ tissues. Western blot was performed to reveal the Ser473-phosphorylated Akt and total Akt levels. Results: HOTAIR was found to be up-regulated in higher grades of AEJ tissues compared to low grades and/or noncancerous tissues. pAkt expression was also found to be up-regulated in tissues of higher tumor stages. We found that the overexpression of HOTAIR finely correlated with elevated Ser473-phosphorylated Akt levels. Conclusion: Upregulated HOTAIR was associated with abnormal activated PI3K/Akt pathway, which might serve as a promising therapeutic strategy for AEJ treatment.

  1. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    PubMed Central

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  2. Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia

    PubMed Central

    Serrano-López, Juana; Serrano, Josefina; Figueroa, Vianihuini; Torres-Gomez, Antonio; Tabares, Salvador; Casaño, Javier; Fernandez-Escalada, Noemi; Sánchez-Garcia, Joaquín

    2013-01-01

    Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia. PMID:23812937

  3. Polyphenol-enriched diet prevents coronary endothelial dysfunction by activating the Akt/eNOS pathway.

    PubMed

    Vilahur, Gemma; Padró, Teresa; Casaní, Laura; Mendieta, Guiomar; López, José A; Streitenberger, Sergio; Badimon, Lina

    2015-03-01

    The Mediterranean diet, rich in polyphenols, has shown to be cardioprotective. However the mechanisms involved remain unknown. We investigated whether supplementation with a pomegranate extract rich in polyphenols renders beneficial effects on coronary function in a clinically relevant experimental model and characterized the underlying mechanisms. Pigs were fed a 10-day normocholesterolemic or hypercholesterolemic diet. Half of the animals were given a supplement of 625 mg/day of a pomegranate extract (Pomanox; 200 mg punicalagins/day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide-synthase inhibitor) were measured using flow Doppler. Akt/endothelial nitric oxide-synthase axis activation, monocyte chemoattractant protein-1 expression, oxidative deoxyribonucleic acid damage in the coronary artery, and lipoprotein resistance to oxidation were evaluated. In dyslipidemic animals, Pomanox supplementation prevented diet-induced impairment of endothelial relaxation, reaching vasodilatory values comparable to normocholesterolemic animals upon stimulation with acetylcholine and/or calcium ionophore. These beneficial effects were associated with vascular Akt/endothelial nitric oxide-synthase activation and lower monocyte chemoattractant protein-1 expression. Pomanox supplementation reduced systemic oxidative stress (higher high-density lipoprotein-antioxidant capacity and higher low-density lipoprotein resistance to oxidation) and coronary deoxyribonucleic acid damage. Normocholesterolemic animals elicited similar drug-related vasodilation regardless of Pomanox supplementation. All animals displayed a similar vasodilatory response to sodium nitroprusside and L-NG-monomethylarginine blunted all vasorelaxation responses except for sodium nitroprusside. Pomanox supplementation hinders hyperlipemia-induced coronary endothelial dysfunction by activating

  4. DCC functions as an accelerator of thalamocortical axonal growth downstream of spontaneous thalamic activity

    PubMed Central

    Castillo-Paterna, Mar; Moreno-Juan, Verónica; Filipchuk, Anton; Rodríguez-Malmierca, Luis; Susín, Rafael; López-Bendito, Guillermina

    2015-01-01

    Controlling the axon growth rate is fundamental when establishing brain connections. Using the thalamocortical system as a model, we previously showed that spontaneous calcium activity influences the growth rate of thalamocortical axons by regulating the transcription of Robo1 through an NF-κB-binding site in its promoter. Robo1 acts as a brake on the growth of thalamocortical axons in vivo. Here, we have identified the Netrin-1 receptor DCC as an accelerator for thalamic axon growth. Dcc transcription is regulated by spontaneous calcium activity in thalamocortical neurons and activating DCC signaling restores normal axon growth in electrically silenced neurons. Moreover, we identified an AP-1-binding site in the Dcc promoter that is crucial for the activity-dependent regulation of this gene. In summary, we have identified the Dcc gene as a novel downstream target of spontaneous calcium activity involved in axon growth. Together with our previous data, we demonstrate a mechanism to control axon growth that relies on the activity-dependent regulation of two functionally opposed receptors, Robo1 and DCC. These two proteins establish a tight and efficient means to regulate activity-guided axon growth in order to correctly establish neuronal connections during development. PMID:25947198

  5. Bamboo leaf extract ameliorates diabetic nephropathy through activating the AKT signaling pathway in rats.

    PubMed

    Ying, Changjiang; Mao, Yizhen; Chen, Lei; Wang, Shanshan; Ling, Hongwei; Li, Wei; Zhou, Xiaoyan

    2017-03-27

    Diabetic nephropathy (DN) is one of the most severe diabetic complication and it is becoming become a worldwide epidemic, accounting for approximately one-third of all case of end-stage renal disease. However, the underlying mechanism and strategy to alleviate renal injury remain unclear. In the present study, we assessed the protective effect of bamboo leaf extract on the DN, and investigated the underlying mechanism by which bamboo leaf extract ameliorating DN. Diabetic rats were induced by 4 weeks high sugar and high fat diet, and then injected a single dose of STZ (35mg/kg) into abdominal cavity. Different dose of bamboo extract (50mg/kg, 100mg/kg and 200mg/kg) were orally administered every day for a period of 12 weeks. Body weight, blood glucose, glycosylated hemoglobin A1c (HbAlc), blood urea nitrogen (BUN), serum creatinine (Scr), and 24-hour urinary protein (24 h-UP) were assessed. Total superoxide dismutase (T-SOD) activity and MDA (methane dicarboxylic aldehyde, MDA) level were tested by assay kit. Microstructural changes were observed by hematoxylin-eosin (HE) staining and electron microscopy. Expression of phosphorylated ser/thr protein kinase (P-AKT), phosphorylated glycogen synthase kinase-3 beta (P-GSK-3β), B cell lymphoma/leukemia 2-associated X protein (BAX) and cleaved-cysteinyl aspartate-specific proteinase-3 (Cleaved Caspase-3) were measured by Western-Blotting (WB). Results showed that diabetic rats had weight loss, high blood glucose, HbAlc, BUN, Scr and 24-UP and T-SOD activity were increased and MDA level was decreased in diabetic rats. Moreover, hyperglycemia could injury renal tissue ultrastructure, inhibit P-AKT level and increase P-GSK-3β, BAX and Cleaved Caspase-3 levels in rats. However, bamboo leaf extract treatment could reduce body weight loss, BUN, Scr, 24 h-UP and MDA level, improve T-SOD activity and alleviate renal injury in diabetic rats. Furthermore, bamboo leaf extract increased P-AKT level, decreased P-GSK-3β, BAX and

  6. Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

    SciTech Connect

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I.; Figueroa, Carlos D.; González, Carlos B.

    2013-11-29

    Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

  7. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells

    PubMed Central

    Liu, Ta-Ming; Ling, Yonghua; Woyach, Jennifer A.; Beckwith, Kyle; Yeh, Yuh-Ying; Hertlein, Erin; Zhang, Xiaoli; Lehman, Amy; Awan, Farrukh; Jones, Jeffrey A.; Andritsos, Leslie A.; Maddocks, Kami; MacMurray, Jessica; Salunke, Santosh B.; Chen, Ching-Shih; Phelps, Mitch A.; Byrd, John C.

    2015-01-01

    Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL. PMID:25293770

  8. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells.

    PubMed

    Liu, Ta-Ming; Ling, Yonghua; Woyach, Jennifer A; Beckwith, Kyle; Yeh, Yuh-Ying; Hertlein, Erin; Zhang, Xiaoli; Lehman, Amy; Awan, Farrukh; Jones, Jeffrey A; Andritsos, Leslie A; Maddocks, Kami; MacMurray, Jessica; Salunke, Santosh B; Chen, Ching-Shih; Phelps, Mitch A; Byrd, John C; Johnson, Amy J

    2015-01-08

    Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

  9. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation

    PubMed Central

    Covarrubias, Anthony J; Aksoylar, Halil Ibrahim; Yu, Jiujiu; Snyder, Nathaniel W; Worth, Andrew J; Iyer, Shankar S; Wang, Jiawei; Ben-Sahra, Issam; Byles, Vanessa; Polynne-Stapornkul, Tiffany; Espinosa, Erika C; Lamming, Dudley; Manning, Brendan D; Zhang, Yijing; Blair, Ian A; Horng, Tiffany

    2016-01-01

    Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation. DOI: http://dx.doi.org/10.7554/eLife.11612.001 PMID:26894960

  10. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.

    PubMed

    Covarrubias, Anthony J; Aksoylar, Halil Ibrahim; Yu, Jiujiu; Snyder, Nathaniel W; Worth, Andrew J; Iyer, Shankar S; Wang, Jiawei; Ben-Sahra, Issam; Byles, Vanessa; Polynne-Stapornkul, Tiffany; Espinosa, Erika C; Lamming, Dudley; Manning, Brendan D; Zhang, Yijing; Blair, Ian A; Horng, Tiffany

    2016-02-19

    Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation.

  11. The novel IGF-IR/Akt-dependent anticancer activities of glucosamine.

    PubMed

    Song, Ki-Hoon; Kang, Ju-Hee; Woo, Jong-Kyu; Nam, Jeong-Seok; Min, Hye-Young; Lee, Ho-Young; Kim, Soo-Youl; Oh, Seung-Hyun

    2014-01-20

    Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway. We used various in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism. We found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress. Taken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer.

  12. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  13. Suppression of isoproterenol-induced apoptosis in H9c2 cardiomyoblast cells by daidzein through activation of Akt.

    PubMed

    Hu, Wei-Syun; Lin, Yueh-Min; Kuo, Wei-Wen; Pan, Lung-Fa; Yeh, Yu-Lan; Li, Yi-Hui; Kuo, Chia-Hua; Chen, Ray-Jade; Padma, V. Vijaya; Chen, Tung-Sheng; Huang, Chih-Yang

    2016-12-31

    Increased serum norepinephrine level is one of pathological processes relating to heart disease (HD). Estrogens are considered as potential therapeutics for the treatment of HD; however, estrogen supplementation shows some side-effects, such as increasing the risk of developing breast, endometrial and ovarian cancers. This study investigated the cardio-protective effects of daidzein (Dai), a selective estrogen receptor modulator (SERM) from soy bean extract, in H9c2 cardiomyoblast cells treated with isoproterenol (ISO), a norepinephrine analog. In this in vitro model, H9c2 cells treated with Dai at different concentrations showed no statistical difference in cell viability. TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) data and western blotting results indicated that Dai treated-H9c2 cells recovered from the damage induced by ISO. The recovery effects of Dai on ISO-induced damage were blocked by inhibition of Akt activation through adding Akt inhibitor. On the other hand, the fold changes of phosphorylated Akt (p-Akt)/Akt normalized with the control for con, 0.25, 0.5, 1, 3 and 24 h of treatment were 1, 2, 5, 13, 11 and 10, respectively. In conclusion, Dai ameliorates apoptosis of cardiomyoblasts induced by ISO through Akt signaling pathway.

  14. Oxidative stress induces proliferation of colorectal cancer cells by inhibiting RUNX3 and activating the Akt signaling pathway.

    PubMed

    Kang, Kyoung Ah; Kim, Ki Cheon; Bae, Suk Chul; Hyun, Jin Won

    2013-11-01

    We recently reported that the tumor suppressor Runt-related transcription factor 3 (RUNX3) is silenced in colorectal cancer cells via oxidative stress-induced hypermethylation of its promoter. The resulting downregulation of RUNX3 expression influences cell proliferation. Activation of the Akt signaling pathway is also associated with cell survival and proliferation; however, the effects of oxidative stress on the relationship between RUNX3 and Akt signaling are largely unknown. Therefore, this study investigated the mechanisms involved in cell proliferation caused by oxidative stress-induced silencing of RUNX3. The levels of RUNX3 mRNA and protein were downregulated in response to treatment of the human colorectal cancer cell line SNU-407 with H2O2. Treatment of the cells with H2O2 also upregulated Akt mRNA and protein expression, and inhibited the binding of RUNX3 to the Akt promoter. The inverse correlation between the expression levels of RUNX3 and Akt in H2O2-treated cells was also associated with nuclear translocation of β-catenin and upregulation of cyclin D1 expression, which induced cell proliferation. H2O2 treatment also increased the binding of β-catenin to the cyclin D1 promoter. The results presented here demonstrate that reactive oxygen species silence the tumor suppressor RUNX3, enhance the Akt-mediated signaling pathway, and promote the proliferation of colorectal cancer cells.

  15. AKT and AMPK activation after high-fat and high-glucose in vitro treatment of prostate epithelial cells.

    PubMed

    Ribeiro, D L; Góes, R M; Pinto-Fochi, M E; Taboga, S R; Abrahamsson, P-A; Dizeyi, N

    2014-06-01

    Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 μM) and/or glucose (450 mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 μM of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate. © Georg Thieme Verlag KG Stuttgart · New York.

  16. Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

    PubMed Central

    Zhai, Bo; Hu, Fengli; Yan, Haijiang; Zhao, Dali; Jin, Xin; Fang, Taishi; Pan, Shangha; Sun, Xueying; Xu, Lishan

    2015-01-01

    Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC. PMID:26381511

  17. Angiotensin II Signaling in Human Preadipose Cells: Participation of ERK1,2-Dependent Modulation of Akt

    PubMed Central

    Dünner, Natalia; Quezada, Carolina; Berndt, F. Andrés; Cánovas, José; Rojas, Cecilia V.

    2013-01-01

    The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK1,2 activities. In the absence of angiotensin II, inhibition of ERK1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK1 protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood. PMID:24098385

  18. HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

    PubMed Central

    Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

    2016-01-01

    AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

  19. All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

    PubMed Central

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F.; Pak, Kwang; Hemmings, Brian A.; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear. PMID:25811375

  20. All Akt isoforms (Akt1, Akt2, Akt3) are involved in normal hearing, but only Akt2 and Akt3 are involved in auditory hair cell survival in the mammalian inner ear.

    PubMed

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F; Pak, Kwang; Hemmings, Brian A; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear-especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.

  1. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    SciTech Connect

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  2. Obesity Increases Vascular Senescence and Susceptibility to Ischemic Injury Through Chronic Activation of Akt and mTOR

    PubMed Central

    Wang, Chao-Yung; Kim, Hyung-Hwan; Hiroi, Yukio; Sawada, Naoki; Salomone, Salvatore; Benjamin, Laura E.; Walsh, Kenneth; Moskowitz, Michael A.; Liao, James K.

    2009-01-01

    Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR)–rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease. PMID:19293429

  3. Coexpression of hyperactivated AKT1 with additional genes activated in leukemia drives hematopoietic progenitor cells to cell cycle block and apoptosis.

    PubMed

    Tang, Yanjuan; Halvarsson, Camilla; Nordigården, Amanda; Kumar, Komal; Åhsberg, Josefine; Rörby, Emma; Wong, Wan Man; Jönsson, Jan-Ingvar

    2015-07-01

    The phosphatidylinositol 3-kinase/AKT pathway is an integral component of signaling involved in the development of many cancers, including myeloid leukemias such as chronic myeloid leukemia and acute myeloid leukemia (AML). Increased AKT1 activity is frequently seen in AML patients, providing leukemic cells with growth and survival promoting signals. An important aspect of AKT1 function is its involvement in cellular metabolism and energy production. Under some circumstances, strong activation of AKT1 increases oxidative stress, which can cause apoptosis when cells progressively build up excess free radicals. This has been described in hematopoietic cells overexpressing activated AKT1; however, whether this is true in cells coexpressing other genetic events involved in leukemia is not known. This prompted us to investigate the effect of constitutively active AKT1 (myristoylated AKT1) in hematopoietic progenitor cells expressing constitutively active signal transducer and activator of transcription 5, Fms-related tyrosine kinase 3-internal tandem duplication, or antiapoptotic B-cell lymphoma 2. Surprisingly, myristoylated AKT1 was incompatible with proliferation driven by both signal transducer and activator of transcription 5 and Fms-related tyrosine kinase 3-internal tandem duplication, which triggered cell cycle block and apoptosis. Moreover, transplantable cells of B-cell lymphoma 2-transgenic mice were impaired in their engraftment ability to recipient mice when expressing hyperactivated AKT1. This was linked to AKT1-mediated proapoptotic functions and not to impairment in homing to the bone marrow. Although cells expressing hyperactivated AKT1 displayed higher levels of reactive oxygen species both in vitro and in vivo, the addition of the antioxidant N-acetyl-L-cysteine significantly reduced apoptosis. Taken together, the results indicate that constitutive AKT1 activity is incompatible with growth- and survival-promoting ability of other activated genes in

  4. Activity of methane oxidizing bacteria along the River Elbe downstream to its estuary

    NASA Astrophysics Data System (ADS)

    Matoušu, A.; Šimek, K.; Bussmann, I.

    2012-04-01

    Contribution of rivers and estuarine systems to the global methane (CH4) budget is still not clear yet and for improving the estimations on the CH4 emission it is essential to cover the whole natural system in large-scale studies. In this context we quantified the activity of methane oxidizing bacteria (as a microbial "biofilter") over almost two seasons along a large European river system, the river Elbe, from its source in the Czech Republic towards to its estuary in the North Sea. Determination of methane oxidation rate was measured using a tritiated CH4 radiotracer technique. Methane concentrations in the water column were analyzed by the headspace method. The range of CH4concentrations and related microbial oxidation activities displayed a strong increase from the upper river parts, which includes mainly natural river, to the polluted downstream canalized parts of the river with high CH4concentrations and microbial activities, where anaerobic sediments occur. In the water column of the estuarine zone a sharp decrease of the CH4 concentration- and CH4 oxidation rates is mainly influenced by the increasing salinity. Additional parameters (temperature, dissolved oxygen, amount of suspended particular matter and nutrient content) are possible factors influencing the methanotrophic activity. Further investigations will compare the population structure of MOB at the different sites along the whole transect.

  5. Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy.

    PubMed

    Nandi, Nilay; Tyra, Lauren K; Stenesen, Drew; Krämer, Helmut

    2014-10-27

    How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type-specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn(S641,731D) or the caspase-resistant Acn(D527A), exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.

  6. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways

    SciTech Connect

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. - Highlights: • CHIP is significantly upregulated in thyroid cancer cells. • Overexpression of CHIP facilitates proliferation and tumorigenesis of thyroid cancer cells. • Silencing of CHIP inhibits the proliferation and tumorigenesis of thyroid cancer cells. • CHIP promotes thyroid cancer cell proliferation via activating the MAPK and AKT pathways.

  7. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  8. α-Lipoic acid interaction with dopamine D2 receptor-dependent activation of the Akt/GSK-3β signaling pathway induced by antipsychotics: potential relevance for the treatment of schizophrenia.

    PubMed

    Deslauriers, Jessica; Desmarais, Christian; Sarret, Philippe; Grignon, Sylvain

    2013-05-01

    Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.

  9. Spatholobus suberectus Exhibits Antidiabetic Activity In Vitro and In Vivo through Activation of AKT-AMPK Pathway.

    PubMed

    Zhao, Peijun; Alam, Md Badrul; Lee, Seok-Hyun; Kim, Young-Jun; Lee, Seul; An, Hongyan; Choi, Hee-Jeong; Son, Hyeong-U; Park, Chul-Hong; Kim, Hyo-Hyun; Lee, Sang-Han

    2017-01-01

    Glucose deposition in peripheral tissue is an important parameter for the treatment of type 2 diabetes mellitus. The aim of this study was to investigate the effects of Spatholobus suberectus (Ss) on glucose disposal in skeletal muscle cells and additionally explore its in vivo antidiabetic potential. Treatment of ethanolic extract of S. suberectus (EeSs) significantly enhanced the glucose uptake, mediated through the enhanced expression of GLUT4 in skeletal muscle via the stimulation of AKT and AMPK pathways in C2C12 cells. Moreover, EeSs have potential inhibitory action on α-glucosidase activity and significantly lowered the postprandial blood glucose levels in STZ-induced diabetic mice, associated with increased expression of GLUT4 and AKT and/or AMPK-mediated signaling cascade in skeletal muscle. Furthermore, administration of EeSs significantly boosted up the antioxidant enzyme expression and also mitigated the gluconeogenesis enzyme such as PEPCK and G-6-Pase enzyme expression in liver tissue of STZ-induced diabetic mice model. Collectively, these findings suggest that EeSs have a high potentiality to mitigate diabetic symptoms through stimulating glucose uptake in peripheral tissue via the activation of AKT and AMPK signaling cascade and augmenting antioxidant potentiality as well as blocking the gluconeogenesis process in diabetic mice.

  10. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  11. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background.

    PubMed

    Davies, Barry R; Greenwood, Hannah; Dudley, Phillippa; Crafter, Claire; Yu, De-Hua; Zhang, Jingchuan; Li, Jing; Gao, Beirong; Ji, Qunsheng; Maynard, Juliana; Ricketts, Sally-Ann; Cross, Darren; Cosulich, Sabina; Chresta, Christine C; Page, Ken; Yates, James; Lane, Clare; Watson, Rebecca; Luke, Richard; Ogilvie, Donald; Pass, Martin

    2012-04-01

    AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2(+) breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials.

  12. Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

    PubMed Central

    Niu, Haixia; Chen, Xue; Gruppo, Ralph A.; Li, Ding; Wang, Yanhua; Zhang, Lin; Wang, Kemin; Chai, Weiran; Sun, Yueping; Ding, Zhongren; Gartner, T. Kent; Liu, Junling

    2012-01-01

    Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets. PMID:23082158

  13. N-methylhemeanthidine chloride, a novel Amaryllidaceae alkaloid, inhibits pancreatic cancer cell proliferation via down-regulating AKT activation

    SciTech Connect

    Guo, Guoli; Yao, Guangmin; Zhan, Guanqun; Hu, Yufeng; Yue, Ming; Cheng, Ling; Liu, Yaping; Ye, Qi; Qing, Guoliang; Zhang, Yonghui; Liu, Hudan

    2014-11-01

    We previously reported the isolation of a novel Amaryllidaceae alkaloid, N-methylhemeanthidine chloride (NMHC), from Zephyranthes candida, which exhibits potent cytotoxicity in a spectrum of tumor cells. However, the mechanism of action remains unclear. Using multiple cell lines derived from human pancreatic cancer, one of the most mortal and refractory human malignancies, we further studied the NMHC-mediated cytotoxicity and found that it induced drastic cytotoxicity in pancreatic cancer cells whereas an insignificant effect on a noncancerous cell line. The NMHC-mediated growth inhibition was more severe than the first-line chemotherapeutic agent gemcitabine, leading to cell cycle arrest, apoptotic death and decreased glycolysis. NMHC exerted its function through down-regulating AKT activation, and the ectopic expression of activated AKT rescued the growth inhibition. Consistently, NMHC injections in a pancreatic cancer xenograft model manifested the anti-tumor effect in vivo. Engrafted tumor cells underwent AKT attenuation and apoptotic death upon treatments. As such, we here demonstrate the AKT inhibition may be one of the mechanisms by which NMHC decreases tumor cell survival rate in vitro and in vivo. Our data thereby suggest that NMHC holds great promise as a potent chemotherapeutic agent against pancreatic cancer and sheds new light on obtaining such agents from natural products toward therapeutic purposes. - Highlights: • N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid. • NMHC exhibits potent anti-neoplastic activity. • NMHC leads to cell cycle arrest, apoptotic death and decreased metabolism. • NMHC down-regulates the AKT signaling pathway.

  14. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes

    PubMed Central

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-01

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP. PMID:28065941

  15. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes.

    PubMed

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-09

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP.

  16. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    PubMed Central

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels. PMID:26682007

  17. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells.

    PubMed

    Hou, Ya-Qin; Yao, Yao; Bao, Yong-Li; Song, Zhen-Bo; Yang, Cheng; Gao, Xiu-Li; Zhang, Wen-Jing; Sun, Lu-Guo; Yu, Chun-Lei; Huang, Yan-Xin; Wang, Guan-Nan; Li, Yu-Xin

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels.

  18. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca(2+)) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca(2+)) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca(2+) concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca(2+) concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca(2+) could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  19. 2-DEOXY-GLUCOSE DOWN REGULATES ENDOTHELIAL AKT AND ERK VIA INTERFERENCE WITH N-LINKED GLYCOSYLATION, INDUCTION OF ENDOPLASMIC RETICULUM STRESS AND GSK-3β ACTIVATION

    PubMed Central

    Kovács, Krisztina; Decatur, Christina; Toro, Marcela; Pham, Dien G.; Liu, Huaping; Jing, Yuqi; Murray, Timothy G.; Lampidis, Theodore J.; Merchan, Jaime R.

    2015-01-01

    Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analog, 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared to tumor cells, but with time dependent, endothelial selective inhibition of Akt and Erk phosphorylation. Down regulation of these critical survival pathways is shown to be due to 2-DG’s interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK-3β activation and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD 105 +) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress and Erk/Akt down regulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents. PMID:26637370

  20. SZ-685C, a marine anthraquinone, is a potent inducer of apoptosis with anticancer activity by suppression of the Akt/FOXO pathway

    PubMed Central

    Xie, Gui'e; Zhu, Xun; Li, Qing; Gu, Minghui; He, Zhenjian; Wu, Jueheng; Li, Jun; Lin, Yongcheng; Li, Mengfeng; She, Zhigang; Yuan, Jie

    2010-01-01

    Background and purpose: The aims of this study were to investigate the anti-cancer activity of SZ-685C, an anthracycline analogue isolated from marine-derived mangrove endophytic fungi, and to explore the molecular mechanisms underlying such activity. Experimental approach: The effect of SZ-685C on the viability of cancer cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SZ-685C-induced apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay and analysis of caspase activation. The effect of SZ-685C on the Akt/FOXO pathway was studied using Western blotting analysis, and the in vivo anti-tumour efficacy was examined in an MDA-MB-435 breast cancer xenograft model. Key results: SZ-685C suppressed the proliferation of six cancer cell lines derived from human breast cancer, prostate cancer, glioma and hepatoma (IC50 values ranged from 3.0 to 9.6 µM) and the growth of breast cancer xenografts in mice. SZ-685C had a direct apoptosis-inducing effect through both the extrinsic and intrinsic apoptotic pathways, as shown by activation of caspase-8 and 9 as well as effector caspase-3 and poly (ADP-ribose) polymerase. Phosphorylation of Akt and its downstream effectors, forkhead box protein O1 and forkhead box protein O3a, was down-regulated in SZ-685C-treated cancer cells. Furthermore, the pro-apoptotic protein Bim was up-regulated by SZ-685C treatment consistent with FOXO dephosphorylation. Conclusions and implications: SZ-685C could induce apoptosis through the Akt/FOXO pathway, which consequently leads to the observed anti-tumour effect both in vitro and in vivo. Our data suggest that SZ-685C may be a potentially promising Akt inhibitor and anti-cancer drug candidate. PMID:20128807

  1. Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wang, Guoliang; Ma, Ning; Meng, Liukun; Wei, Yingjie; Gui, Jingang

    2015-12-01

    Over-activated PI3K/Akt signaling, a pathway strongly related to cancer survival and proliferation, has been reported recently to be involved in pulmonary artery smooth muscle cell apoptosis and proliferation in pulmonary hypertension (PH). In this study, we observed greatly increased lipocalin-2 (Lcn2) expression accompanied with over-activated PI3K/Akt signaling in a standard rat model of PH induced by monocrotaline. In view of the close relationship between Lcn2 and PI3K/Akt pathway, we hypothesized that the up-regulated Lcn2 might be a trigger of over-activated PI3K/Akt signaling in PH. Our results showed that Lcn2 significantly activated the PI3K/Akt pathway (determined by augmented Akt phosphorylation and up-regulated Mdm2) and significantly promoted proliferation (assessed by Ki67 staining) in cultured human pulmonary artery smooth muscle cells. Furthermore, we demonstrated that inhibition of Akt phosphorylation (LY294002) abrogated the Lcn2-promoted proliferation in cultured human pulmonary artery smooth muscle cells. In conclusion, Lcn2 significantly promoted human pulmonary artery smooth muscle cell proliferation by activating PI3K/Akt pathway. Further study on the role and mechanism of Lcn2 will help explore novel therapeutic strategies based on attenuating over-activated PI3K/Akt signaling in PH.

  2. Complex formation of p65/RelA with nuclear Akt1 for enhanced transcriptional activation of NF-{kappa}B

    SciTech Connect

    Kwon, Osong; Kim, Kyung A; He, Long; Jung, Mira; Jeong, Sook Jung; Ahn, Jong Seog Kim, Bo Yeon

    2008-01-25

    Akt1 was revealed to interact with Ki-Ras in the cytoplasm of Ki-Ras-transformed human prostate epithelial cells, 267B1/K-ras. Moreover, p65/RelA in the nucleus was found to interact with both Ki-Ras and Akt1, suggesting the nuclear translocation of Akt1:Ki-Ras complex for NF- {kappa}B activation. In support of this, compared with wild type Akt1, the dominant negative Akt1 mutant was decreased in its nuclear expression, reducing the Ki-Ras-induced NF-{kappa}B transcriptional activation. Moreover, inhibitors of Ras (sulindac sulfide and farnesyltransferase inhibitor I) or PI3K/Akt (wortmannin), reduced the amounts of Akt1 and Ki-Ras in the nucleus as well as partial NF-{kappa}B activity. The complete inhibition of Ki-Ras-induced NF-{kappa}B activation, however, could only be obtained by combined treatment with wortmannin and proteasome inhibitor-1. Accordingly, clonogenic assay showed Akt1 contribution to I{kappa}B{alpha}-mediated NF-{kappa}B activation for oncogenic cell growth by Ki-Ras. Our data suggest a crucial role of Ki-Ras:Akt1 complex in NF-{kappa}B transcriptional activation and enhancement of cell survival.

  3. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma.

    PubMed

    Watanabe, Tadashi; Nakamura, Shigeo; Ono, Toshiya; Ui, Sadaharu; Yagi, Syota; Kagawa, Hiroki; Watanabe, Hisami; Ohe, Tomoyuki; Mashino, Tadahiko; Fujimuro, Masahiro

    2014-08-15

    Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin's B-cell lymphoma and is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1',1'-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated whether pyrrolidinium fullerene in combination with the HSP90 inhibitor (geldanamycin; GA) or valproate, potentiated the cytotoxic effects on PEL cells. Compared to treatment with pyrrolidinium fullerene alone, the addition of low-concentration GA or valproate enhanced the cytotoxic activity of pyrrolidinium fullerene. These results indicate that pyrrolidinium fullerene could be used as a novel therapy for the treatment of PEL

  4. Dysfunctional MnSOD leads to redox dysregulation and activation of prosurvival AKT signaling in uterine leiomyomas

    PubMed Central

    Vidimar, Vania; Gius, David; Chakravarti, Debabrata; Bulun, Serdar E.; Wei, Jian-Jun; Kim, J. Julie

    2016-01-01

    AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM’s prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬ PMID:27847869

  5. MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics.

    PubMed

    Masters, Thomas A; Tumbarello, David A; Chibalina, Margarita V; Buss, Folma

    2017-06-06

    APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma

    SciTech Connect

    Watanabe, Tadashi; Nakamura, Shigeo; Ono, Toshiya; Ui, Sadaharu; Yagi, Syota; Kagawa, Hiroki; Watanabe, Hisami; Ohe, Tomoyuki; Mashino, Tadahiko; Fujimuro, Masahiro

    2014-08-15

    Highlights: • Seven fullerenes were evaluated in terms of their cytotoxic effects on B-lymphomas. • Pyrrolidinium fullerene induced apoptosis of KSHV-infected B-lymphoma PEL cells. • The activation of Akt is essential for PEL cell survival. • Pyrrolidinium fullerene activated caspase-9 by inactivating Akt in PEL cells. • Pyrrolidinium fullerene have potential as novel drugs for the treatment of PEL. - Abstract: Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin’s B-cell lymphoma and is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated

  7. A Small Molecule Inhibits Akt through Direct Binding to Akt and Preventing Akt Membrane Translocation*

    PubMed Central

    Kim, Donghwa; Sun, Mei; He, Lili; Zhou, Qing-Hua; Chen, Jun; Sun, Xia-Meng; Bepler, Gerold; Sebti, Said M.; Cheng, Jin Q.

    2010-01-01

    The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)308 and Ser(P)473) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt. PMID:20068047

  8. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    PubMed

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

  9. Modification of Caffeic Acid with Pyrrolidine Enhances Antioxidant Ability by Activating AKT/HO-1 Pathway in Heart

    PubMed Central

    Ku, Hui-Chun; Lee, Shih-Yi; Yang, Kai-Chien; Kuo, Yueh-Hsiung; Su, Ming-Jai

    2016-01-01

    Overproduction of free radicals during ischemia/reperfusion (I/R) injury leads to an interest in using antioxidant therapy. Activating an endogenous antioxidant signaling pathway is more important due to the fact that the free radical scavenging behavior in vitro does not always correlate with a cytoprotection effect in vivo. Caffeic acid (CA), an antioxidant, is a major phenolic constituent in nature. Pyrrolidinyl caffeamide (PLCA), a derivative of CA, was compared with CA for their antioxidant and cytoprotective effects. Our results indicate that CA and PLCA exert the same ability to scavenge DPPH in vitro. In response to myocardial I/R stress, PLCA was shown to attenuate lipid peroxydation and troponin release more than CA. These responses were accompanied with a prominent elevation in AKT and HO-1 expression and a preservation of mnSOD expression and catalase activity. PLCA also improved cell viability and alleviated the intracellular ROS level more than CA in cardiomyocytes exposed to H2O2. When inhibiting the AKT or HO-1 pathways, PLCA lost its ability to recover mnSOD expression and catalase activity to counteract with oxidative stress, suggesting AKT/HO-1 pathway activation by PLCA plays an important role. In addition, inhibition of AKT signaling further abolished HO-1 activity, while inhibition of HO-1 signaling attenuated AKT expression, indicating cross-talk between the AKT and HO-1 pathways. These protective effects may contribute to the cardiac function improvement by PLCA. These findings provide new insight into therapeutic approaches using a modified natural compound against oxidative stress from myocardial injuries. PMID:26845693

  10. Rituximab activates Syk and AKT in CD20-positive B cell lymphoma cells dependent on cell membrane cholesterol levels.

    PubMed

    Nozaki, Yumi; Mitsumori, Toru; Yamamoto, Takeo; Kawashima, Ichiro; Shobu, Yuki; Hamanaka, Satoshi; Nakajima, Kei; Komatsu, Norio; Kirito, Keita

    2013-08-01

    The introduction of rituximab, an anti-CD20 monoclonal antibody, has dramatically improved the treatment outcomes of patients with B cell lymphoma. Nevertheless, the clinical response to rituximab varies, and a subpopulation of patients does not respond well to this antibody. Although several molecular events have been shown to be involved in the mechanism of action of rituximab, recent studies have demonstrated that intracellular signaling pathways and the direct effects of rituximab on cell membrane components are responsible for the antilymphoma action of this drug. In the present study, we demonstrated that rituximab activated Syk and Akt, molecules with antiapoptotic functions, in several CD20-positive lymphoma cell lines. Notably, rituximab activated Syk and Akt in all the tested primary lymphoma samples from six patients. Our results show that the cholesterol levels in lymphoma cell membranes have a crucial role in the regulation of Syk and Akt. The depletion of cholesterol from the cell membrane completely blocked rituximab-induced Syk and Akt activation. Simvastatin, an inhibitor of cholesterol synthesis, also abrogated rituximab-mediated Syk and Akt activation. Finally, we report that rituximab inhibited the apoptosis induced by chemotherapeutic drugs, which was observed solely in Akt-activated cells. This work demonstrates for the first time that rituximab paradoxically works to suppress apoptosis under certain conditions in a manner that is dependent on the cell membrane cholesterol level. Our observations provide novel insights and suggest that the cell membrane cholesterol level represents a new biomarker for predicting patient response to rituximab. Furthermore, the modulation of lipid rafts could provide a new strategy for enhancing the antilymphoma action of rituximab. Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  11. Aurora-A induces cell survival and chemoresistance by activation of Akt through a p53-dependent manner in ovarian cancer cells.

    PubMed

    Yang, Hua; He, Lili; Kruk, Patricia; Nicosia, Santo V; Cheng, Jin Q

    2006-11-15

    Aurora-A is frequently altered in epithelial malignancies. Overexpressing Aurora-A induces centrosome amplification and G2/M cell cycle progression. We have previously shown elevated level of Aurora-A in ovarian cancer and activation of telomerase by Aurora-A in human mammary and ovarian epithelia. Here we report that Aurora-A protects ovarian cancer cells from apoptosis induced by chemotherapeutic agent and activates Akt pathway in a p53-dependent manner. Ectopic expression of Aurora-A renders cells resistant to cisplatin (CDDP), etoposide and paclitaxel-induced apoptosis and stimulates Akt1 and Akt2 activity in wild-type p53 but not p53-null ovarian cancer cells. Aurora-A inhibits cytochrome C release and Bax conformational change induced by CDDP. Knockdown of Aurora-A by RNAi sensitizes cells to CDDP-induced apoptosis and decreases phospho-Akt level in wild-type p53 cells. Reintroduction of p53 decreases Akt1 and Akt2 activation and restores CDDP sensitivity in p53-null but not p53-null-Aurora-A cells. Inhibition of Akt by small molecule inhibitor, API-2, overcomes the effects of Aurora-A-on cell survival and Bax mitochondrial translocation. Taken collectively, these data indicate that Aurora-A activates Akt and induces chemoresistance in a p53-dependent manner and that inhibition of Akt may be an effective means of overcoming Aurora-A-associated chemoresistance in ovarian cancer cells expressing wild-type p53.

  12. Suppression of AKT expression by miR-153 produced anti-tumor activity in lung cancer.

    PubMed

    Yuan, Ye; Du, Weijie; Wang, Ying; Xu, Chaoqian; Wang, Jinghao; Zhang, Yang; Wang, Huimin; Ju, Jiaming; Zhao, Liang; Wang, Zhiguo; Lu, Yanjie; Cai, Benzhi; Pan, Zhenwei

    2015-03-15

    Lung cancer is one of the leading causes of cancer death worldwide. microRNAs have been shown to be a novel class of regulators in lung cancer. Here, we explored the role of miR-153 in the pathogenesis of lung cancer and its therapeutic potential. miR-153 was significantly decreased in lung cancer tissues than the adjacent tissues. The protein and mRNA levels of protein kinase B (AKT), which were shown to promote tumor growth, were both increased in lung cancer tissues than adjacent tissues. Overexpression of miR-153 significantly inhibited AKT protein expression, which were abrogated by co-transfection of AMO-153, the specific inhibitor of miR-153. Luciferase assay showed that transfection of miR-153 markedly suppressed the fluorescent intensity of chimeric vectors carrying the 3'UTR of AKT1, while produced no effect on the mutant construct, indicating that AKT is regulated by miR-153. Overexpression of miR-153 significantly inhibited the proliferation and migration, and promoted apoptosis of cultured lung cancer cells in vitro, and suppressed the growth of xenograft tumors in vivo. Interestingly, lung cancer cells with lower endogenous miR-153 expression are more sensitive to ectopic overexpressed miR-153. The IC50 of miR-153 on lung cancer cells is positive correlated with the endogenous miR-153 level, while negative correlated with AKT level. Knockdown of AKT expression suppressed lung cancer cell proliferation. In summary, miR-153 exerted anti-tumor activity in lung cancer by targeting on AKT. The sensitivity of lung cancer cells to miR-153 is determined by its endogenous miR-153 level.

  13. Pigment epithelium-derived factor protects cone photoreceptor-derived 661W cells from light damage through Akt activation.

    PubMed

    Rapp, Matthew; Woo, Grace; Al-Ubaidi, Muayyad R; Becerra, S Patricia; Subramanian, Preeti

    2014-01-01

    Pigment epithelium-derived factor (PEDF) can delay and prevent the death of photoreceptors in vivo. We investigated the survival activity of PEDF on cone photoreceptor-derived 661W cells in culture, the presence of PEDF receptor (PEDF-R) in these cells and the activation of prosurvival Akt. Cell death was induced by light exposure in the presence of 9-cis retinal. Cell viability assays showed that PEDF increased the number of 661W cells exposed to these conditions. Western blots showed that PEDF-treated 661W cells had a higher ratio of phosphorylated Akt to total Akt than untreated cells. The PEDF receptor PEDF-R was immunodetected in the plasma membrane fractions of 661W cells. The results demonstrated that PEDF can protect 661W cells against light-induced cell death and suggest that the binding of PEDF to cell surface PEDF-R triggers a prosurvival signaling pathway.

  14. Dystrophin Glycoprotein Complex-associated Gβγ Subunits Activate Phosphatidyl Inositol-3-Kinase/Akt signaling in Skeletal Muscle in a Laminin-dependent Manner

    PubMed Central

    Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W.

    2010-01-01

    Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein, (Gαβγ) to bind, changing the activation state of the Gsα subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gβγ, released when Gsα is activated, is known to bind phosphatidylinositol 3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gβγ, using immunoprecipitation and immunoblotting, and purified Gβγ. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to α-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gβγ also caused PI3K and Akt activation. These results show that DGC-Gβγ is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin β1 compared to normal muscle. This integrin binds laminin, Gβγ, and PI3K. Collectively, these suggest that PI3K is an important target for the Gβγ, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gβγ-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Up-regulating integrin β1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease. PMID:19117013

  15. Dystrophin glycoprotein complex-associated Gbetagamma subunits activate phosphatidylinositol-3-kinase/Akt signaling in skeletal muscle in a laminin-dependent manner.

    PubMed

    Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W

    2009-05-01

    Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein (Galphabetagamma) to bind, changing the activation state of the Gsalpha subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gbetagamma, released when Gsalpha is activated, is known to bind phosphatidylinositol-3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gbetagamma, using immunoprecipitation and immunoblotting, and purified Gbetagamma. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to alpha-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gbetagamma also caused PI3K and Akt activation. These results show that DGC-Gbetagamma is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin beta1 compared to normal muscle. This integrin binds laminin, Gbetagamma, and PI3K. Collectively, these suggest that PI3K is an important target for the Gbetagamma, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gbetagamma-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin beta1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.

  16. Control of macrophage metabolism and activation by mTOR and Akt signaling

    PubMed Central

    Covarrubias, Anthony J.; Aksoylar, H. Ibrahim; Horng, Tiffany

    2015-01-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies. PMID:26360589

  17. Control of macrophage metabolism and activation by mTOR and Akt signaling.

    PubMed

    Covarrubias, Anthony J; Aksoylar, H Ibrahim; Horng, Tiffany

    2015-08-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies.

  18. Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.

    PubMed

    Hellyer, N J; Kim, M S; Koland, J G

    2001-11-09

    The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

  19. EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line

    PubMed Central

    Popeda, Marta; Ksiazkiewicz, Magdalena; Grzela, Dawid P.; Walczak, Maciej P.; Banaszczyk, Mateusz; Peciak, Joanna; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant–EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis. PMID:27153109

  20. Ischemic post-conditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats.

    PubMed

    Xie, Rong; Wang, Peng; Ji, Xunming; Zhao, Heng

    2013-12-01

    While pre-conditioning is induced before stroke onset, ischemic post-conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild-type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild-type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri-infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1-30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase. Post-conditioning did not attenuate infarction in nude rats measured 2 days post-stroke, but improved neurological function in nude rats and reduced brain damage 30 days after stroke. It resulted in increased-activities of Akt and mTOR, S6K and p-4EBP1. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC.

  1. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function.

    PubMed

    Phuchareon, Janyaporn; McCormick, Frank; Eisele, David W; Tetsu, Osamu

    2015-07-21

    Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

  2. A critical role of the small GTPase Rac1 in Akt2-mediated GLUT4 translocation in mouse skeletal muscle.

    PubMed

    Takenaka, Nobuyuki; Izawa, Rumi; Wu, Junyuan; Kitagawa, Kaho; Nihata, Yuma; Hosooka, Tetsuya; Noguchi, Tetsuya; Ogawa, Wataru; Aiba, Atsu; Satoh, Takaya

    2014-03-01

    Insulin promotes glucose uptake in skeletal muscle by inducing the translocation of the glucose transporter GLUT4 to the plasma membrane. The serine/threonine kinase Akt2 has been implicated as a key regulator of this insulin action. However, the mechanisms whereby Akt2 regulates multiple steps of GLUT4 translocation remain incompletely understood. Recently, the small GTPase Rac1 has been identified as a skeletal muscle-specific regulator of insulin-stimulated glucose uptake. Here, we show that Rac1 is a critical downstream component of the Akt2 pathway in mouse skeletal muscle as well as cultured myocytes. GLUT4 translocation induced by constitutively activated Akt2 was totally dependent on the expression of Rac1 in L6 myocytes. Moreover, we observed the activation of Rac1 when constitutively activated Akt2 was ectopically expressed. Constitutively activated Akt2-triggered Rac1 activation was diminished by knockdown of FLJ00068, a guanine nucleotide exchange factor for Rac1. Knockdown of Akt2, on the other hand, markedly reduced Rac1 activation by a constitutively activated mutant of phosphoinositide 3-kinase. In mouse skeletal muscle, constitutively activated mutants of Akt2 and phosphoinositide 3-kinase, when ectopically expressed, induced GLUT4 translocation. Muscle-specific rac1 knockout markedly diminished Akt2- or phosphoinositide 3-kinase-induced GLUT4 translocation, highlighting a crucial role of Rac1 downstream of Akt2. Taken together, these results strongly suggest a novel regulatory link between Akt2 and Rac1 in insulin-dependent signal transduction leading to glucose uptake in skeletal muscle.

  3. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  4. Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis.

    PubMed

    Mancini, Maria L; Lien, Evan C; Toker, Alex

    2016-04-05

    One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K).

  5. Aβ-Induced Drp1 phosphorylation through Akt activation promotes excessive mitochondrial fission leading to neuronal apoptosis.

    PubMed

    Kim, Dah Ihm; Lee, Ki Hoon; Gabr, Amr Ahmed; Choi, Gee Euhn; Kim, Jun Sung; Ko, So Hee; Han, Ho Jae

    2016-11-01

    Mitochondrial dysfunction is known as one of causative factors in Alzheimer's disease (AD), inducing neuronal cell death. Mitochondria regulate their functions through changing their morphology. The present work was undertaken to investigate whether Amyloid β (Aβ) affects mitochondrial morphology in neuronal cells to induce apoptosis. Aβ treatment induced not only the fragmentation of mitochondria but also neuronal apoptosis in association with an increase in caspase-9 and -3 activity. Calcium influx induced by Aβ up-regulated the activation of Akt through CaMKII resulting in changes to the phosphorylation level of Drp1 in a time-dependent manner. Translocation of Drp1 from the cytosol to mitochondria was blocked by CB-124005 (an Akt inhibitor). Recruitment of Drp1 to mitochondria led to ROS generation and mitochondrial fission, accompanied by dysfunction of mitochondria such as loss of membrane potential and ATP production. ROS generation and mitochondrial dysfunction by Aβ were attenuated when treated with Mdivi-1, a selective Drp1 inhibitor. Furthermore, the sustained Akt activation induced not only the fragmentation of mitochondria but also the activation of mTOR, eventually suppressing autophagy. Inhibition of autophagic clearance of Aβ led to increased ROS levels and aggravating mitochondrial defects, which were blocked by Rapamycin (an mTOR inhibitor). In conclusion, sustained phosphorylation of Akt by Aβ directly activates Drp1 and inhibits autophagy through the mTOR pathway. Together, these changes elicit abundant mitochondrial fragmentation resulting in ROS-mediated neuronal apoptosis.

  6. P2X7 receptors stimulate AKT phosphorylation in astrocytes

    PubMed Central

    Jacques-Silva, Maria C; Rodnight, Richard; Lenz, Guido; Liao, Zhongji; Kong, Qiongman; Tran, Minh; Kang, Yuan; Gonzalez, Fernando A; Weisman, Gary A; Neary, Joseph T

    2004-01-01

    Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′-triphosphate (UTP) and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism. PMID:15023862

  7. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    PubMed

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The anti-inflammatory effect of 3-deoxysappanchalcone is mediated by inducing heme oxygenase-1 via activating the AKT/mTOR pathway in murine macrophages.

    PubMed

    Kim, Jun-Hyeong; Choo, Young-Yeon; Tae, Nara; Min, Byung-Sun; Lee, Jeong-Hyung

    2014-10-01

    3-Deoxysappanchalcone (3-DSC), isolated from Caesalpinia sappan (Leguminosae), is a chalcone that exerts a variety of pharmacological activities. In the present study, we demonstrated that 3-DSC exerts anti-inflammatory activity in murine macrophages by inducing heme oxygenase-1 (HO-1) expression at the translational level. Treatment of RAW264.7 cells with 3-DSC induced HO-1 protein expression in a dose- and time-dependent manner without affecting HO-1 mRNA expression. Mitogen-activated protein kinase inhibitors or actinomycin D, a transcriptional inhibitor, did not block 3-DSC-mediated HO-1 induction. However, 3-DSC-mediated HO-1 induction was completely blocked by treatment with cycloheximide, a translational inhibitor, or rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Strikingly, 3-DSC increased the phosphorylation level of mTOR downstream target molecules such as eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1), as well as AKT in a dose- and time-dependent manner, suggesting that the 3-DSC induces HO-1 expression by activating the AKT/mTOR pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, 3-DSC inhibited the production of nitric oxide (NO) and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Inhibition of HO-1 activity by treatment with tin protoporphyrin IX, a specific HO-1 inhibitor, abrogated the inhibitory effects of 3-DSC on the production of NO and IL-6 in LPS-stimulated RAW264.7 cells. Taken together, 3-DSC may be an effective HO-1 inducer at the translational level that has anti-inflammatory effects, and a valuable compound for modulating inflammatory conditions.

  9. Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

    PubMed Central

    2011-01-01

    Background Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Methods Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Results Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher

  10. Aspirin enhances the cytotoxic activity of bortezomib against myeloma cells via suppression of Bcl-2, survivin and phosphorylation of AKT

    PubMed Central

    Ding, Jiang-Hua; Yuan, Li-Ya; Chen, Guo-An

    2017-01-01

    In our previous study, it was found that aspirin (ASA) exerted antimyeloma actions in vivo and in vitro. The resistance to bortezomib (BTZ) in multiple myeloma (MM) is partly due to AKT activation and the upregulation of survivin induced by BTZ, which are the targets of ASA in gastric and ovarian cancer, respectively. Thus, the present study investigated the interaction between ASA and BTZ in MM and further clarified the underlying mechanisms. MM1.S and RPMI-8226 cell lines harboring the N- and K-Ras mutations, respectively, were treated with 2.5 mM ASA, 10 nM BTZ and ASA+BTZ for different durations. The proliferation and apoptosis of the cells were determined, and the underlying mechanisms governing the interaction of ASA and BTZ were examined in the MM cells. Treatment with ASA+BTZ caused higher rates of proliferative inhibition and apoptosis in the MM1.S and RPMI-8226 cells in time-dependent manner, compared with either agent alone. A drug interaction assay revealed the additive effect of ASA and BTZ on the myeloma cells. ASA alone inhibited the levels of phosphorylated AKT (p-AKT) and survivin, whereas BTZ alone augmented the levels of p-AKT and survivin. Of note, ASA markedly decreased the upregulation of p-AKT and survivin induced by BTZ. Treatment with ASA+BTZ significantly suppressed the level of Bcl-2, compared with either agent alone. ASA may potentiate the antimyeloma activity of BTZ against myeloma cells via suppression of AKT phosphorylation, survivin and Bcl-2, indicating the potential of ASA+BTZ in treating MM, particularly for cases of BTZ-refractory/relapsed MM. PMID:28356941

  11. SG2NA enhances cancer cell survival by stabilizing DJ-1 and thus activating Akt

    SciTech Connect

    Tanti, Goutam Kumar Pandey, Shweta; Goswami, Shyamal K.

    2015-08-07

    SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth. - Highlights: • Reactive oxygen species (ROS) play potential role in cancer cell proliferation. • It enhances the association between DJ-1 and Akt mediated by SG2NA. • In cancer cells SG2NA stabilizes DJ-1 by inhibiting it from proteosomal degradation. • DJ-1 then activates Akt and cancer cells get their property of enhanced proliferation by sustained activation of Akt. • Further study on this field could lead to new target for cancer therapy.

  12. Selumetinib Attenuates Skeletal Muscle Wasting in Murine Cachexia Model through ERK Inhibition and AKT Activation.

    PubMed

    Quan-Jun, Yang; Yan, Huo; Yong-Long, Han; Li-Li, Wan; Jie, Li; Jin-Lu, Huang; Jin, Lu; Peng-Guo, Chen; Run, Gan; Cheng, Guo

    2017-02-01

    Cancer cachexia is a multifactorial syndrome affecting the skeletal muscle. Previous clinical trials showed that treatment with MEK inhibitor selumetinib resulted in skeletal muscle anabolism. However, it is conflicting that MAPK/ERK pathway controls the mass of the skeletal muscle. The current study investigated the therapeutic effect and mechanisms of selumetinib in amelioration of cancer cachexia. The classical cancer cachexia model was established via transplantation of CT26 colon adenocarcinoma cells into BALB/c mice. The effect of selumetinib on body weight, tumor growth, skeletal muscle, food intake, serum proinflammatory cytokines, E3 ligases, and MEK/ERK-related pathways was analyzed. Two independent experiments showed that 30 mg/kg/d selumetinib prevented the loss of body weight in murine cachexia mice. Muscle wasting was attenuated and the expression of E3 ligases, MuRF1 and Fbx32, was inhibited following selumetinib treatment of the gastrocnemius muscle. Furthermore, selumetinib efficiently reduced tumor burden without influencing the cancer cell proliferation, cumulative food intake, and serum cytokines. These results indicated that the role of selumetinib in attenuating muscle wasting was independent of cancer burden. Detailed analysis of the mechanism revealed AKT and mTOR were activated, while ERK, FoxO3a, and GSK3β were inhibited in the selumetinib -treated cachexia group. These indicated that selumetinib effectively prevented skeletal muscle wasting in cancer cachexia model through ERK inhibition and AKT activation in gastrocnemius muscle via cross-inhibition. The study not only elucidated the mechanism of MEK/ERK inhibition in skeletal muscle anabolism, but also validated selumetinib therapy as an effective intervention against cancer cachexia. Mol Cancer Ther; 16(2); 334-43. ©2016 AACR.

  13. PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells.

    PubMed

    Klein, Benjamin Y; Tamir, Hadassah; Welch, Martha G

    2011-11-01

    Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5  nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8  nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5  nM OT for 30  min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.

  14. FAM83B-mediated activation of PI3K/AKT and MAPK signaling cooperates to promote epithelial cell transformation and resistance to targeted therapies

    PubMed Central

    Cipriano, Rocky; Miskimen, Kristy L.S.; Bryson, Benjamin L.; Foy, Chase R.; Bartel, Courtney A.; Jackson, Mark W.

    2013-01-01

    Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers. PMID:23676467

  15. Inhibition of constitutively activated phosphoinositide 3-kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1-defective breast cancer cells.

    PubMed

    Yi, Yong Weon; Kang, Hyo Jin; Kim, Hee Jeong; Hwang, Jae Seok; Wang, Antai; Bae, Insoo

    2013-09-01

    Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103, and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.

  16. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

    SciTech Connect

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio

    2014-07-18

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

  17. Galectin-3 protects keratinocytes from UVB-induced apoptosis by enhancing AKT activation and suppressing ERK activation

    PubMed Central

    Saegusa, Jun; Hsu, Daniel K.; Liu, Wei; Kuwabara, Ichiro; Kuwabara, Yasuko; Yu, Lan; Liu, Fu-Tong

    2009-01-01

    Keratinocytes undergo apoptosis in a variety of physiological and pathological conditions. Galectin-3 is a member of a family of β-galactoside-binding animal lectins expressed abundantly in keratinocytes and other epithelial cells. Here we have studied the regulatory role of galectin-3 in keratinocyte apoptosis by using cells from gene-targeted galectin-3 null (gal3−/−) mice. We showed that galectin-3 mRNA was transiently upregulated in ultraviolet-B (UVB)-irradiated wild-type keratinocytes. We found that gal3−/− keratinocytes were significantly more sensitive to apoptosis induced by UVB as well as various other stimuli, both in vitro and in vivo, than wild-type cells. Moreover, we demonstrated that increased apoptosis in gal3−/− keratinocytes was attributable to higher extracellular signal-regulated kinase (ERK) activation and lower AKT activation after UVB irradiation. We conclude that endogenous galectin-3 is an anti-apoptotic molecule in keratinocytes functioning by suppressing ERK activation and enhancing AKT activation and may play a role in the development of apoptosis-related skin diseases. PMID:18463681

  18. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  19. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  20. FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

    PubMed

    Hayashi, Hiroki; Nakagami, Hironori; Takami, Yoichi; Koriyama, Hiroshi; Mori, Masaki; Tamai, Katsuto; Sun, Jianxin; Nagao, Kaori; Morishita, Ryuichi; Kaneda, Yasufumi

    2009-06-01

    In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

  1. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    PubMed Central

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  2. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    NASA Astrophysics Data System (ADS)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  3. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    SciTech Connect

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-06

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  4. Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo via inactivation of Akt and activation of JNK

    PubMed Central

    Budhraja, Amit; Gao, Ning; Zhang, Zhuo; Son, Young-Ok; Cheng, Senping; Wang, Xin; Ding, Songze; Hitron, Andrew; Chen, Gang; Luo, Jia; Shi, Xianglin

    2015-01-01

    In this study, we investigated the functional role of Akt and JNK signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin-induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 down-regulation, cytochrome c release from mitochondria and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspases activation, and apoptosis. Conversely, LY294002 and a dominant negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of JNK pathway showed marked reduction in apigenin-induced caspases activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. PMID:22084167

  5. mTOR Promotes Survival and Astrocytic Characteristics Induced by Pten/Akt Signaling in Glioblastoma1

    PubMed Central

    Hu, Xiaoyi; Pandolfi, Pier Paolo; Li, Yi; Koutcher, Jason A; Rosenblum, Marc; Holland, Eric C

    2005-01-01

    Abstract Combined activation of Ras and Akt leads to the formation of astrocytic glioblastoma multiforme (GBM) in mice. In human GBMs, AKT is not mutated but is activated in approximately 70% of these tumors, in association with loss of PTEN and/or activation of receptor tyrosine kinases. Mechanistic justification for the therapeutic blockade of targets downstream of AKT, such as mTOR, in these cancers requires demonstration that the oncogenic effect of PTEN loss is through elevated AKT activity. We demonstrate here that loss of Pten is similar to Akt activation in the context of glioma formation in mice. We further delineate the role of mTOR activity downstream of Akt in the maintenance of Akt+KRas-induced GBMs. Blockade of mTOR results in regional apoptosis in these tumors and conversion in the character of surviving tumor cells from astrocytoma to oligodendroglioma. These data suggest that mTOR activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells. Furthermore, our study provides the first example of conversion between two distinct tumor types usually thought of as belonging to specific lineages, and provides evidence for signal transduction-mediated transdifferentiation between glioma subtypes. PMID:15967113

  6. Akt pathway activation and increased neuropeptide Y mRNA expression in the rat hippocampus: implications for seizure blockade.

    PubMed

    Goto, Eduardo M; Silva, Marcelo de Paula; Perosa, Sandra R; Argañaraz, Gustavo A; Pesquero, João B; Cavalheiro, Esper A; Naffah-Mazzacoratti, Maria G; Teixeira, Vicente P C; Silva, José A

    2010-04-01

    The aim of this study was to analyze the expression of survival-related molecules such Akt and integrin-linked kinase (ILK) to evaluate Akt pathway activation in epileptogenesis process. Furthermore, was also investigated the mRNA expression of neuropeptide Y, a considered antiepileptic neuropeptide, in the pilocarpine-induced epilepsy. Male Wistar rats were submitted to the pilocarpine model of epilepsy. Hippocampi were removed 6h (acute phase), 12h (late acute), 5d (silent) and 60d (chronic) after status epilepticus (SE) onset, and from animals that received pilocarpine but did not develop SE (partial group). Hippocampi collected were used to specify mRNA expression using Real-Time PCR. Immunohistochemistry assay was employed to place ILK distribution in the hippocampus and Western blot technique was used to determine Akt activation level. A decrease in ILK mRNA content was found during acute (0.39+/-0.03) and chronic (0.48+/-0.06) periods when compared to control group (0.87+/-0.10). Protein levels of ILK were also diminished during both periods. Partial group showed increased ILK mRNA expression (0.80+/-0.06) when compared with animals in the acute stage. Silent group had ILK mRNA and immunoreactivity similar to control group. Western blot assay showed an augmentation in Akt activation in silent period (0.52+/-0.03) in comparison with control group (0.44+/-0.01). Neuropeptide Y mRNA expression increased in the partial group (1.67+/-0.22) and in the silent phase (1.45+/-0.29) when compared to control group (0.36+/-0.12). Results suggest that neuropeptide Y (as anticonvulsant) might act in protective mechanisms occurred during epileptic phenomena. Together with ILK expression and Akt activation, these molecules could be involved in hippocampal neuroprotection in epilepsy. Copyright 2009 Elsevier Ltd. All rights reserved.

  7. Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation

    PubMed Central

    Yang, Changjun; Talukder, M.A. Hassan; Varadharaj, Saradhadevi; Velayutham, Murugesan; Zweier, Jay L.

    2013-01-01

    Aims The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC. Methods and results Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOSSer1176, AktSer473, and PKAThr197 phosphorylation was increased following IPC. I/R decreased eNOSSer1176 phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOSSer1176 phosphorylation and quantitative Western blots showed ∼24% modification of eNOSSer1176 following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased AktSer473 or PKAThr197 phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOSSer1176 phosphorylation as well as cardioprotection. Conclusion Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role. PMID:22977010

  8. Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

    PubMed Central

    MacDonald, Elizabeth M.; Andres-Mateos, Eva; Mejias, Rebeca; Simmers, Jessica L.; Mi, Ruifa; Park, Jae-Sung; Ying, Stephanie; Hoke, Ahmet; Lee, Se-Jin; Cohn, Ronald D.

    2014-01-01

    The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy. PMID:24504412

  9. N-methylhemeanthidine chloride, a novel Amaryllidaceae alkaloid, inhibits pancreatic cancer cell proliferation via down-regulating AKT activation.

    PubMed

    Guo, Guoli; Yao, Guangmin; Zhan, Guanqun; Hu, Yufeng; Yue, Ming; Cheng, Ling; Liu, Yaping; Ye, Qi; Qing, Guoliang; Zhang, Yonghui; Liu, Hudan

    2014-11-01

    We previously reported the isolation of a novel Amaryllidaceae alkaloid, N-methylhemeanthidine chloride (NMHC), from Zephyranthes candida, which exhibits potent cytotoxicity in a spectrum of tumor cells. However, the mechanism of action remains unclear. Using multiple cell lines derived from human pancreatic cancer, one of the most mortal and refractory human malignancies, we further studied the NMHC-mediated cytotoxicity and found that it induced drastic cytotoxicity in pancreatic cancer cells whereas an insignificant effect on a noncancerous cell line. The NMHC-mediated growth inhibition was more severe than the first-line chemotherapeutic agent gemcitabine, leading to cell cycle arrest, apoptotic death and decreased glycolysis. NMHC exerted its function through down-regulating AKT activation, and the ectopic expression of activated AKT rescued the growth inhibition. Consistently, NMHC injections in a pancreatic cancer xenograft model manifested the anti-tumor effect in vivo. Engrafted tumor cells underwent AKT attenuation and apoptotic death upon treatments. As such, we here demonstrate the AKT inhibition may be one of the mechanisms by which NMHC decreases tumor cell survival rate in vitro and in vivo. Our data thereby suggest that NMHC holds great promise as a potent chemotherapeutic agent against pancreatic cancer and sheds new light on obtaining such agents from natural products toward therapeutic purposes.

  10. TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

    PubMed Central

    Hill, Richard; Madureira, Patricia A.; Ferreira, Bibiana; Baptista, Inês; Machado, Susana; Colaço, Laura; dos Santos, Marta; Liu, Ningshu; Dopazo, Ana; Ugurel, Selma; Adrienn, Angyal; Kiss-Toth, Endre; Isbilen, Murat; Gure, Ali O.; Link, Wolfgang

    2017-01-01

    Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells. PMID:28276427

  11. CX3CR1-Mediated Akt1 Activation Contributes to the Paclitaxel-Induced Painful Peripheral Neuropathy in Rats.

    PubMed

    Li, Dai; Chen, Hui; Luo, Xiao-Huan; Sun, Yang; Xia, Wei; Xiong, Yuan-Chang

    2016-06-01

    Painful peripheral neuropathy is a serious dose-limiting side effect of paclitaxel therapy, which unfortunately often happens during the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms of the painful peripheral neuropathy remain largely unknown. Here, we found that paclitaxel treatment (3 × 8 mg/kg, cumulative dose 24 mg/kg) upregulated the expression of CX3CR1 and phosphorylated Akt1 in DRG and spinal dorsal horn. Blocking of Akt1 pathway activation with different inhibitor (MK-2206 or LY294002) significantly attenuated mechanical allodynia and thermal hyperalgesia induced by paclitaxel. Furthermore, inhibition of CX3CR1 by using neutralizing antibody not only prevented Akt1 activation in DRG and spinal dorsal horn but also alleviated pain-related behavior induced by paclitaxel treatment. This study suggested that CX3CR1/Akt1 signaling pathway may be a potential target for prevention and reversion of the painful peripheral neuropathy induced by paclitaxel.

  12. mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

    PubMed Central

    Palmieri, Michela; Pal, Rituraj; Nelvagal, Hemanth R.; Lotfi, Parisa; Stinnett, Gary R.; Seymour, Michelle L.; Chaudhury, Arindam; Bajaj, Lakshya; Bondar, Vitaliy V.; Bremner, Laura; Saleem, Usama; Tse, Dennis Y.; Sanagasetti, Deepthi; Wu, Samuel M.; Neilson, Joel R.; Pereira, Fred A.; Pautler, Robia G.; Rodney, George G.; Cooper, Jonathan D.; Sardiello, Marco

    2017-01-01

    Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases. PMID:28165011

  13. Erbin loss promotes cancer cell proliferation through feedback activation of Akt-Skp2-p27 signaling

    SciTech Connect

    Huang, Hao; Song, Yuhua; Wu, Yan; Guo, Ning; Ma, Yuanfang; Qian, Lu

    2015-07-31

    Erbin localizes at the basolateral membrane to regulate cell junctions and polarity in epithelial cells. Dysregulation of Erbin has been implicated in tumorigenesis, and yet it is still unclear if and how disrupted Erbin regulates the biological behavior of cancer cells. We report here that depletion of Erbin leads to cancer cell excessive proliferation in vitro and in vivo. Erbin deficiency accelerates S-phase entry by down-regulating CDK inhibitors p21 and p27 via two independent mechanisms. Mechanistically, Erbin loss promotes p27 degradation by enhancing E3 ligase Skp2 activity though augmenting Akt signaling. Interestingly, we also show that Erbin is an unstable protein when the Akt-Skp2 signaling is aberrantly activated, which can be specifically destructed by SCF-Skp2 ligase. Erbin loss facilitates cell proliferation and migration in Skp2-dependent manner. Thus, our finding illustrates a novel negative feedback loop between Erbin and Akt-Skp2 signaling. It suggests disrupted Erbin links polarity loss, hyperproliferation and tumorigenesis. - Highlights: • Erbin loss leads to cancer cell excessive proliferation in vitro and in vivo. • Erbin loss accelerates cell cycle though down-regulating p21 and p27 expression. • Erbin is a novel negative modulator of Akt1-Skp2-p27 signaling pathway. • Our study suggests that Erbin loss contributes to Skp2 oncogenic function.

  14. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    PubMed

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt.

  15. Hyperforin Inhibits Akt1 Kinase Activity and Promotes Caspase-Mediated Apoptosis Involving Bad and Noxa Activation in Human Myeloid Tumor Cells

    PubMed Central

    Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

    2011-01-01

    Background The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. Methodology and Results HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Significance Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment. PMID:21998731

  16. Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

    PubMed Central

    Joh, Eun-Ha; Hollenbaugh, Joseph A.; Kim, Baek; Kim, Dong-Hyun

    2012-01-01

    While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor. PMID:23189201

  17. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

    SciTech Connect

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-08

    Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

  18. A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

    PubMed Central

    Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

    2017-01-01

    Background Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Materials and Methods Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. Results In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. Conclusions These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts. PMID:28045948

  19. A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK.

    PubMed

    Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

    2017-01-01

    Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts.

  20. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    PubMed

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-06

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.

  1. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue*

    PubMed Central

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J.

    2016-01-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr308 and Ser473 upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser473-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  2. Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways.

    PubMed

    El Omri, Abdelfatteh; Han, Junkyu; Kawada, Kiyokazu; Ben Abdrabbah, Manef; Isoda, Hiroko

    2012-02-09

    Luteolin, a 3', 4', 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

  3. Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway

    PubMed Central

    Mu, Qingchun; Wang, Lijun; Yu, Fengbo; Gao, Haijun; Lei, Ting; Li, Peiwen; Liu, Pengfei; Zheng, Xu; Hu, Xitong; Chen, Yong; Jiang, Zhenfeng; Sayari, Arash J; Shen, Jia; Huang, Haiyan

    2015-01-01

    Glioblastomas multiforme (GBM) are the most frequently occurring malignant brain cancers. Treatment for GBM consists of surgical resection and subsequent adjuvant radiation therapy and chemotherapy. Despite this, GBM patient survival is limited to 12–15 months, and researchers are continually trying to develop improved therapy options. Insulin-like growth factor 2 mRNA-binding protein 2 (Imp2) is known to be upregulated in many cancers and is known to regulate the signaling activity of insulin-like growth factor 2 (IGF2). However, relatively little is known about its role in malignant development of GBM. In this study, we first found Imp2 is upregulated in GBM tissues by using clinical samples and public database search. Studies with loss and gain of Imp2 expression in in vitro GBM cell culture system demonstrated the role of Imp2 in promoting GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT). Additionally, our results show that Imp2 regulates the activity of IGF2, which further activates PI3K/Akt signaling, thereby to promote GBM malignancy. Inhibition of Imp2 was also found to sensitize GBM to temozolomide treatment. These observations add to the current knowledge of GBM biology, and may prove useful in development of more effective GBM therapy. PMID:25719943

  4. Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway.

    PubMed

    Mu, Qingchun; Wang, Lijun; Yu, Fengbo; Gao, Haijun; Lei, Ting; Li, Peiwen; Liu, Pengfei; Zheng, Xu; Hu, Xitong; Chen, Yong; Jiang, Zhenfeng; Sayari, Arash J; Shen, Jia; Huang, Haiyan

    2015-01-01

    Glioblastomas multiforme (GBM) are the most frequently occurring malignant brain cancers. Treatment for GBM consists of surgical resection and subsequent adjuvant radiation therapy and chemotherapy. Despite this, GBM patient survival is limited to 12-15 months, and researchers are continually trying to develop improved therapy options. Insulin-like growth factor 2 mRNA-binding protein 2 (Imp2) is known to be upregulated in many cancers and is known to regulate the signaling activity of insulin-like growth factor 2 (IGF2). However, relatively little is known about its role in malignant development of GBM. In this study, we first found Imp2 is upregulated in GBM tissues by using clinical samples and public database search. Studies with loss and gain of Imp2 expression in in vitro GBM cell culture system demonstrated the role of Imp2 in promoting GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT). Additionally, our results show that Imp2 regulates the activity of IGF2, which further activates PI3K/Akt signaling, thereby to promote GBM malignancy. Inhibition of Imp2 was also found to sensitize GBM to temozolomide treatment. These observations add to the current knowledge of GBM biology, and may prove useful in development of more effective GBM therapy.

  5. A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death

    PubMed Central

    Frías, Alex; Lambies, Guillem; Viñas-Castells, Rosa; Martínez-Guillamon, Catalina; Dave, Natàlia

    2015-01-01

    Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor β1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3β (GSK-3β) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3β phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the β-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3β inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3β inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism. PMID:26711268

  6. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling

    PubMed Central

    Zhang, P-F; Li, K-S; Shen, Y-h; Gao, P-T; Dong, Z-R; Cai, J-B; Zhang, C; Huang, X-Y; Tian, M-X; Hu, Z-Q; Gao, D-M; Fan, J; Ke, A-W; Shi, G-M

    2016-01-01

    Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial–mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment. PMID:27100895

  7. Syndecan-2 Attenuates Radiation-induced Pulmonary Fibrosis and Inhibits Fibroblast Activation by Regulating PI3K/Akt/ROCK Pathway via CD148.

    PubMed

    Tsoyi, Konstantin; Chu, Sarah G; Patino-Jaramillo, Nasly G; Wilder, Julie; Villalba, Julian; Doyle-Eisele, Melanie; McDonald, Jacob; Liu, Xiaoli; El-Chemaly, Souheil; Perrella, Mark A; Rosas, Ivan O

    2017-09-08

    Radiation-induced pulmonary fibrosis is a severe complication of patients treated with thoracic irradiation. We have previously shown that syndecan-2 reduces fibrosis by exerting alveolar epithelial cytoprotective effects. Here, we investigate whether syndecan-2 attenuates radiation-induced pulmonary fibrosis by inhibiting fibroblast activation. C57BL/6 wild type (WT) mice and transgenic (TG) mice that overexpress human syndecan-2 in alveolar macrophages were exposed to 14 Gy whole thoracic radiation. Twenty-four weeks after irradiation, lungs were collected for histological, protein and mRNA evaluation of pulmonary fibrosis, profibrotic gene expression and alpha-smooth muscle actin (α-SMA) expression. Mouse lung fibroblasts were activated with TGF-β1 in the presence or absence of syndecan-2. Cell proliferation, migration and gel contraction were assessed at different time points. Irradiation resulted in significantly increased mortality and pulmonary fibrosis in WT mice that was associated with elevated lung expression of transforming growth factor-beta1 (TGF-β1) downstream target genes and cell death compared to irradiated syndecan-2 TG mice. In mouse lung fibroblasts, syndecan-2 inhibited α-SMA expression, cell contraction, proliferation and migration induced by TGF-β1. Syndecan-2 attenuated PI3K/Akt/ROCK signaling and serum response factor (SRF) binding to the α-SMA promoter. Syndecan-2 attenuates pulmonary fibrosis in mice exposed to radiation and inhibits TGF-β1-induced fibroblast-myofibroblast differentiation, migration and proliferation by downregulating PI3K/Akt/ROCK signaling and blocking SRF binding to the α-SMA promoter via CD148. These findings suggest that syndecan-2 has potential as an antifibrotic therapy in radiation-induced lung fibrosis.

  8. Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway.

    PubMed

    Yeramian, Andree; Sorolla, Anabel; Velasco, Ana; Santacana, Maria; Dolcet, Xavier; Valls, Joan; Abal, Leandre; Moreno, Sara; Egido, Ramón; Casanova, Josep M; Puig, Susana; Vilella, Ramón; Llombart-Cussac, Antonio; Matias-Guiu, Xavier; Martí, Rosa M

    2012-02-15

    Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit. Copyright © 2011 UICC.

  9. Tyrosines-740/751 of PDGFRβ contribute to the activation of Akt/Hif1α/TGFβ nexus to drive high glucose-induced glomerular mesangial cell hypertrophy.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2017-09-23

    Glomerular mesangial cell hypertrophy contributes to the complications of diabetic nephropathy. The mechanism by which high glucose induces mesangial cell hypertrophy is poorly understood. Here we explored the role of the platelet-derived growth factor receptor-β (PDGFRβ) tyrosine kinase in driving the high glucose-induced mesangial cell hypertrophy. We show that high glucose stimulates the association of the PDGFRβ with PI 3 kinase leading to tyrosine phosphorylation of the latter. High glucose-induced Akt kinase activation was also dependent upon PDGFRβ and its tyrosine phosphorylation at 740/751 residues. Inhibition of PDGFRβ activity, its downregulation and expression of its phospho-deficient (Y740/751F) mutant inhibited mesangial cell hypertrophy by high glucose. Interestingly, expression of constitutively active Akt reversed this inhibition, indicating a role of Akt kinase downstream of PDGFRβ phosphorylation in this process. The transcription factor Hif1α is a target of Akt kinase. siRNAs against Hif1α inhibited the high glucose-induced mesangial cell hypertrophy. In contrast, increased expression of Hif1α induced hypertrophy similar to high glucose. We found that inhibition of PDGFRβ and expression of PDGFRβ Y740/751F mutant significantly inhibited the high glucose-induced expression of Hif1α. Importantly, expression of Hif1α countered the inhibition of mesangial cell hypertrophy induced by siPDGFRβ or PDGFRβ Y740/751F mutant. Finally, we show that high glucose-stimulated PDGFRβ tyrosine phosphorylation at 740/751 residues and the tyrosine kinase activity of the receptor regulate the transforming growth factor-β (TGFβ) expression by Hif1α. Thus we define the cell surface PDGFRβ as a major link between high glucose and its effectors Hif1α and TGFβ for induction of diabetic mesangial cell hypertrophy. Copyright © 2017. Published by Elsevier Inc.

  10. Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

    PubMed

    Yamaguchi, Kazunori; Takanashi, Tomoka; Nasu, Kentaro; Tamai, Keiichi; Mochizuki, Mai; Satoh, Ikuro; Ine, Shoji; Sasaki, Osamu; Satoh, Kennichi; Tanaka, Nobuyuki; Harigae, Hideo; Sugamura, Kazuo

    2016-05-01

    The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells.

  11. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

    PubMed Central

    Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  12. Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF

    PubMed Central

    Wang, Xusheng; Chen, Haiyan; Tian, Ruiyun; Zhang, Yiling; Drutskaya, Marina S.; Wang, Chengmei; Ge, Jianfeng; Fan, Zhimeng; Kong, Deqiang; Wang, Xiaoxiao; Cai, Ting; Zhou, Ying; Wang, Jingwen; Wang, Jinmei; Wang, Shan; Qin, Zhihai; Jia, Huanhuan; Wu, Yue; Liu, Jia; Nedospasov, Sergei A.; Tredget, Edward E.; Lin, Mei; Liu, Jianjun; Jiang, Yuyang; Wu, Yaojiong

    2017-01-01

    Skin stem cells can regenerate epidermal appendages; however, hair follicles (HF) lost as a result of injury are barely regenerated. Here we show that macrophages in wounds activate HF stem cells, leading to telogen–anagen transition (TAT) around the wound and de novo HF regeneration, mostly through TNF signalling. Both TNF knockout and overexpression attenuate HF neogenesis in wounds, suggesting dose-dependent induction of HF neogenesis by TNF, which is consistent with TNF-induced AKT signalling in epidermal stem cells in vitro. TNF-induced β-catenin accumulation is dependent on AKT but not Wnt signalling. Inhibition of PI3K/AKT blocks depilation-induced HF TAT. Notably, Pten loss in Lgr5+ HF stem cells results in HF TAT independent of injury and promotes HF neogenesis after wounding. Thus, our results suggest that macrophage-TNF-induced AKT/β-catenin signalling in Lgr5+ HF stem cells has a crucial role in promoting HF cycling and neogenesis after wounding. PMID:28345588

  13. Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas.

    PubMed

    Yamaguchi, Hiroshi; Kuboki, Yuko; Hatori, Takashi; Yamamoto, Masakazu; Shiratori, Keiko; Kawamura, Shunji; Kobayashi, Makio; Shimizu, Michio; Ban, Shinichi; Koyama, Isamu; Higashi, Morihiro; Shin, Nobuhiro; Ishida, Kazuyuki; Morikawa, Takanori; Motoi, Fuyuhiko; Unno, Michiaki; Kanno, Atsushi; Satoh, Kennichi; Shimosegawa, Tooru; Orikasa, Hideki; Watanabe, Tomoo; Nishimura, Kazuhiko; Harada, Youji; Furukawa, Toru

    2011-12-01

    Intraductal tubulopapillary neoplasm (ITPN) is a recently recognized rare variant of intraductal neoplasms of the pancreas. Molecular aberrations underlying the neoplasm remain unknown. We investigated somatic mutations in PIK3CA, PTEN, AKT1, KRAS, and BRAF. We also investigated aberrant expressions of phosphorylated AKT, phosphatase and tensin homolog (PTEN), tumor protein 53 (TP53), SMAD4, and CTNNB1 in 11 cases of ITPNs and compared these data with those of 50 cases of intraductal papillary mucinous neoplasm (IPMN), another distinct variant of pancreatic intraductal neoplasms. Mutations in PIK3CA were found in 3 of 11 ITPNs but not in IPMNs (P = 0.005; Fisher exact test). In contrast, mutations in KRAS were found in none of the ITPNs but were found in 26 of the 50 IPMNs (P = 0.001; Fisher exact test). PIK3CA mutations were associated with strong expression of phosphorylated AKT (P < 0.001; the Mann-Whitney U test). Moreover, the expression of phosphorylated AKT was apparent in most ITPNs but only in a few IPMNs (P < 0.001; the Mann-Whitney U test). Aberrant expressions of TP53, SMAD4, and CTNNB1 were not statistically different between these neoplasms. Mutations in PIK3CA and the expression of phosphorylated AKT were not associated with age, sex, tissue invasion, and patients' prognosis in ITPNs. These results indicate that activation of the phosphatidylinositol 3-kinase pathway may play a crucial role in ITPNs but not in IPMNs. In contrast, the mutation in KRAS seems to play a major role in IPMNs but not in ITPNs. The activated phosphatidylinositol 3-kinase pathway may be a potential target for molecular diagnosis and therapy of ITPNs.

  14. AG and UAG induce β-casein expression via activation of ERK1/2 and AKT pathways

    PubMed Central

    Li, Sunan; Liu, Juxiong; Lv, Qingkang; Zhang, Chuan; Xu, Shiyao; Yang, Dongxue; Huang, Bingxu; Zeng, Yalong; Gao, Yingjie

    2016-01-01

    Abstract The ghrelin peptides were found to circulate in two major forms: acylated ghrelin (AG) and unacylated ghrelin (UAG). Previous studies showed that AG regulates β-casein (CSN2) expression in mammary epithelial cells. However, little is known about the mechanisms by which AG regulates CSN2 gene and protein expression. Evidence suggests that UAG has biological activity through GHSR1a-independent mechanisms. Here, we investigated the possible GHSR1a-mediated effect of UAG on the expression of CSN2 in primary bovine mammary epithelial cells (pbMECs) isolated from lactating cow. We found that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in pbMECs in comparison with the control group. Increased expression of CSN2 was blocked by [D-Lys3]-GHRP-6 (an antagonist of the GHSR1a) and NF449 (a Gs-α subunit inhibitor) in pbMECs. In addition, both AG and UAG activated AKT/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, whereas [D-Lys3]-GHRP-6 and NF449 inhibited the phosphorylation of AKT and ERK1/2 in pbMECs respectively. Blockade of ERK1/2 and AKT signaling pathways prevented the expression of CSN2 induced by AG or UAG. Finally, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Taken together, these results demonstrate that both AG and UAG act on ERK1/2 and AKT signaling pathways to facilitate the expression of CSN2 in a GHSR1a-dependent manner. PMID:26873999

  15. Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    PubMed Central

    Zhang, Mei-qi; Zheng, Yue-liang; Chen, Huan; Tu, Jian-feng; Shen, Ye; Guo, Jun-ping; Yang, Xiang-hong; Yuan, Shu-ren; Chen, Liang-zhong; Chai, Jing-jie; Lu, Jian-hong; Zhai, Chang-lin

    2013-01-01

    Aim: To investigate the mechanisms underlying the protective effects of sodium tanshinone IIA sulfonate (STS) in an ischemia-reperfusion (I/R)-induced rat myocardial injury model. Methods: Male SD rats were iv injected with STS, STS+LY294002 or saline (NS) for 15 d. Then the hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function, infarction size and area at risk were assessed. Cell apoptosis was evaluated with TUNEL staining, DNA laddering and measuring caspase-3 activity. In addition, isolated cardiomyocytes of neonatal rats were pretreated with the above drugs, then exposed to H2O2 (200 mol/L) for 1 h. Cell apoptosis was detected using flow cytometric assay. The levels of p-Akt, p-FOXO3A and Bim were examined with immunoblotting. Results: Compared to NS group, administration of STS (20 mg/kg) significantly reduced myocardial infarct size (40.28%±5.36% in STS group vs 59.52%±7.28% in NS group), and improved the myocardial function as demonstrated by the increased values of dp/dtmax, LVDP and coronary flow at different reperfusion time stages. Furthermore, STS significantly decreased the rate of apoptotic cells (15.11%±3.71% in STS group vs 38.21%±7.83% in NS group), and reduced caspase-3 activity to nearly a quarter of that in NS group. Moreover, STS significantly increased the phosphorylation of Akt and its downstream target FOXO3A, and decreased the expression of pro-apoptotic gene Bim. Co-treatment with the PI3K inhibitor LY294002 (40 mg/kg) partially countered the protective effects induced by STS treatment. In isolated cardiomyocytes, STS exerted similar protective effects as shown in the ex vivo I/R model. Conclusion: STS pretreatment reduces infarct size and improves cardiac function in an I/R-induced rat myocardial injury model via activation of Akt/FOXO3A/Bim-mediated signal pathway. PMID:24077633

  16. CYP2J2 and its metabolites (epoxyeicosatrienoic acids) attenuate cardiac hypertrophy by activating AMPKα2 and enhancing nuclear translocation of Akt1.

    PubMed

    Wang, Bei; Zeng, Hesong; Wen, Zheng; Chen, Chen; Wang, Dao Wen

    2016-10-01

    Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5'-AMP-activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p-Akt1), and stimulated nuclear translocation of p-Akt1, to exert their antihypertrophic effects. AMPKα2(-/-) mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2(-/-) mice. The CYP2J2 metabolites, 11,12-EET, activated AMPKα2 to induce nuclear translocation of p-Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co-immunoprecipitation analysis, we found that AMPKα2β2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12-EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  17. FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway.

    PubMed

    Yu, Guanzhen; Zhou, Aidong; Xue, Jianfei; Huang, Chen; Zhang, Xia; Kang, Shin-Hyuk; Chiu, Wen-Tai; Tan, Christina; Xie, Keping; Wang, Jiejun; Huang, Suyun

    2015-05-10

    The autocrine platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) signaling pathway promotes breast cancer tumorigenesis, but the mechanisms for its dysregulation in breast cancer are largely unknown. In the study, we identified PDGF-A as a novel transcriptional target of FoxM1. FoxM1 directly binds to two sites in the promoter of PDGF-A and activates its transcription. Mutation of these FoxM1-binding sites diminished PDGF-A promoter activity. Increased FoxM1 resulted in the upregulation of PDGF-A, which led to activation of the AKT pathway and increased breast cancer cell proliferation and tumorigenesis, whereas knockdown of FoxM1 does the opposite. Blocking AKT activation with a phosphoinositide 3-kinase/AKT inhibitor d