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Sample records for phosphoinositide 3-kinase pathway

  1. Targeting the phosphoinositide 3-kinase (PI3K) pathway in cancer

    PubMed Central

    Liu, Pixu; Cheng, Hailing; Roberts, Thomas M.; Zhao, Jean J.

    2011-01-01

    The phosphoinositide 3-kinase (PI3K) pathway, a critical signal transduction system linking oncogenes and multiple receptor classes to many essential cellular functions, is perhaps the most commonly activated signaling pathway in human cancer. This pathway thus presents both an opportunity and a challenge for cancer therapy. Even as inhibitors that target PI3K isoforms and other major nodes in the pathway including AKT and mTOR reach clinical trials, major issues remain. Here we highlight recent progress made in our understanding of the PI3K pathway and discuss both the promises and challenges for the therapeutic development of agents targeting the PI3K pathway in cancer. PMID:19644473

  2. v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation

    PubMed Central

    Akagi, Tsuyoshi; Shishido, Tomoyuki; Murata, Kazutaka; Hanafusa, Hidesaburo

    2000-01-01

    v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF. PMID:10852971

  3. The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alterations and Therapeutic Implications

    PubMed Central

    Arcaro, Alexandre; Guerreiro, Ana S

    2007-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class IA of PI3K comprises the p110α, p110β and p110δ isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110α, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110α and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110α, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context. PMID:19384426

  4. RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia

    PubMed Central

    Edwards, Holly; Xie, Chengzhi; LaFiura, Katherine M.; Dombkowski, Alan A.; Buck, Steven A.; Boerner, Julie L.; Taub, Jeffrey W.; Matherly, Larry H.

    2009-01-01

    RUNX1 (AML1) encodes the core binding factor α subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the δ catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)–kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease. PMID:19638627

  5. The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions.

    PubMed

    Pauls, Samantha D; Lafarge, Sandrine T; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

  6. The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions

    PubMed Central

    Pauls, Samantha D.; Lafarge, Sandrine T.; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J.

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

  7. Novel roles for class II Phosphoinositide 3-Kinase C2β in signalling pathways involved in prostate cancer cell invasion

    PubMed Central

    Mavrommati, Ioanna; Cisse, Ouma; Falasca, Marco; Maffucci, Tania

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) regulate several cellular functions such as proliferation, growth, survival and migration. The eight PI3K isoforms are grouped into three classes and the three enzymes belonging to the class II subfamily (PI3K-C2α, β and γ) are the least investigated amongst all PI3Ks. Interest on these isoforms has been recently fuelled by the identification of specific physiological roles for class II PI3Ks and by accumulating evidence indicating their involvement in human diseases. While it is now established that these isoforms can regulate distinct cellular functions compared to other PI3Ks, there is still a limited understanding of the signalling pathways that can be specifically regulated by class II PI3Ks. Here we show that PI3K-C2β regulates mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) activation in prostate cancer (PCa) cells. We further demonstrate that MEK/ERK and PI3K-C2β are required for PCa cell invasion but not proliferation. In addition we show that PI3K-C2β but not MEK/ERK regulates PCa cell migration as well as expression of the transcription factor Slug. These data identify novel signalling pathways specifically regulated by PI3K-C2β and they further identify this enzyme as a key regulator of PCa cell migration and invasion. PMID:26983806

  8. Shiga toxin type-2 (Stx2) induces glutamate release via phosphoinositide 3-kinase (PI3K) pathway in murine neurons

    PubMed Central

    Obata, Fumiko; Hippler, Lauren M.; Saha, Progyaparamita; Jandhyala, Dakshina M.; Latinovic, Olga S.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) can cause central nervous system (CNS) damage resulting in paralysis, seizures, and coma. The key STEC virulence factors associated with systemic illness resulting in CNS impairment are Shiga toxins (Stx). While neurons express the Stx receptor globotriaosylceramide (Gb3) in vivo, direct toxicity to neurons by Stx has not been studied. We used murine neonatal neuron cultures to study the interaction of Shiga toxin type 2 (Stx2) with cell surface expressed Gb3. Single molecule imaging three dimensional STochastic Optical Reconstruction Microscopy—Total Internal Reflection Fluorescence (3D STORM-TIRF) allowed visualization and quantification of Stx2-Gb3 interactions. Furthermore, we demonstrate that Stx2 increases neuronal cytosolic Ca2+, and NMDA-receptor inhibition blocks Stx2-induced Ca2+ influx, suggesting that Stx2-mediates glutamate release. Phosphoinositide 3-kinase (PI3K)-specific inhibition by Wortmannin reduces Stx2-induced intracellular Ca2+ indicating that the PI3K signaling pathway may be involved in Stx2-associated glutamate release, and that these pathways may contribute to CNS impairment associated with STEC infection. PMID:26236186

  9. Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica.

    PubMed

    Dowd, Georgina C; Bhalla, Manmeet; Kean, Bernard; Thomas, Rowan; Ireton, Keith

    2016-06-01

    Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-α1, Dock180, focal adhesion kinase (FAK), Grp1, LL5α, LL5β, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-α1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlB-mediated entry of L. monocytogenes Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes. PMID:27068087

  10. Phosphoinositide 3-kinases-a historical perspective.

    PubMed

    Toker, Alex

    2012-01-01

    The phosphoinositide 3-kinase (PI 3-K) signal relay pathway represents arguably one of the most intensely studied mechanisms by which extracellular signals elicit cellular responses through the generation of second messengers that are associated with cell growth and transformation. This chapter reviews the many landmark discoveries in the PI 3-K signaling pathway in biology and disease, from the identification of a novel phosphoinositide kinase activity associated with transforming oncogenes in the 1980s, to the identification of oncogenic mutations in the catalytic subunit of PI 3-K in the mid 2000s. Two and a half decades of intense research have provided clear evidence that the PI 3-K pathway controls virtually all aspects of normal cellular physiology, and that deregulation of one or more proteins that regulate or transduce the PI 3-K signal ultimately leads to human pathology. The most recent efforts have focused on the development of specific PI 3-K inhibitors that are currently being evaluated in clinical trials for a range of disease states.This chapter is devoted to a historical review of the landmark findings in the PI 3-K from its relatively humble beginnings in the early to mid 1980s up until the present day. When considering the key findings in the history of PI 3-K, it is essential to recognize the landmark studies by Lowell and Mabel Hokin in the 1950s who were the first to describe that extracellular agonists such as acetylcholine could stimulate the incorporation of radiolabeled phosphate into phospholipids (Hokin and Hokin 1953). Their work initiated an entirely new field of lipid signaling, and subsequent studies in the 1970s by Michell and Lapetina who linked phosphoinositide turnover to membrane-associated receptors that initiate intracellular calcium mobilization (Lapetina and Michell 1973). Later studies revealed that the phospholipase-mediated breakdown of the same minor membrane phospholipids such as PtdIns-4,5-P(2) (phosphatidylinositol-4

  11. Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

    PubMed

    Ostrakhovitch, Elena A; Lordnejad, Mohammad Reza; Schliess, Freimut; Sies, Helmut; Klotz, Lars-Oliver

    2002-01-15

    Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a

  12. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    PubMed

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  13. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  14. Class II phosphoinositide 3-kinase C2β regulates a novel signaling pathway involved in breast cancer progression

    PubMed Central

    Abbott, Jonathan J.; Piñeiro, Roberto; Buus, Richard; Iezzi, Manuela; Ricci, Francesca; Bergamaschi, Daniele; Ostano, Paola; Chiorino, Giovanna; Lattanzio, Rossano; Broggini, Massimo; Piantelli, Mauro; Maffucci, Tania; Falasca, Marco

    2016-01-01

    It is now well established that the enzymes phosphoinositide 3-kinases (PI3Ks) have a key role in the development and progression of many cancer types and indeed PI3Ks inhibitors are currently being tested in clinical trials. Although eight distinct PI3K isoforms exist, grouped into three classes, most of the evidence currently available are focused on one specific isoform with very little known about the potential role of the other members of this family in cancer. Here we demonstrate that the class II enzyme PI3K-C2β is overexpressed in several human breast cancer cell lines and in human breast cancer specimens. Our data indicate that PI3K-C2β regulates breast cancer cell growth in vitro and in vivo and that PI3K-C2β expression in breast tissues is correlated with the proliferative status of the tumor. Specifically we show that downregulation of PI3K-C2β in breast cancer cell lines reduces colony formation, induces cell cycle arrest and inhibits tumor growth, in particular in an estrogen-dependent in vivo xenograft. Investigation of the mechanism of the PI3K-C2β-dependent regulation of cell cycle progression and cell growth revealed that PI3K-C2β regulates cyclin B1 protein levels through modulation of microRNA miR-449a levels. Our data further demonstrate that downregulation of PI3K-C2β inhibits breast cancer cell invasion in vitro and breast cancer metastasis in vivo. Consistent with this, PI3K-C2β is highly expressed in lymph-nodes metastases compared to matching primary tumors. These data demonstrate that PI3K-C2β plays a pivotal role in breast cancer progression and in metastasis development. Our data indicate that PI3K-C2β may represent a key molecular switch that regulates a rate-limiting step in breast tumor progression and therefore it may be targeted to limit breast cancer spread. PMID:26934321

  15. Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway

    SciTech Connect

    Ogata, Takehiro; Ueyama, Tomomi . E-mail: tueyama@kuhp.kyoto-u.ac.jp; Nomura, Tetsuya; Asada, Satoshi; Tagawa, Masashi; Nakamura, Tomoyuki; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa . E-mail: hidemasa@kuhp.kyoto-u.ac.jp

    2007-07-27

    We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

  16. The phosphoinositide 3-kinase signaling pathway is involved in the control of modified low-density lipoprotein uptake by human macrophages.

    PubMed

    Michael, Daryn R; Davies, Thomas S; Laubertová, Lucia; Gallagher, Hayley; Ramji, Dipak P

    2015-03-01

    The transformation of macrophages into lipid-loaded foam cells is a critical early event in the pathogenesis of atherosclerosis. Both receptor-mediated uptake of modified LDL, mediated primarily by scavenger receptors-A (SR-A) and CD36 along with other proteins such as lipoprotein lipase (LPL), and macropinocytosis contribute to macrophage foam cell formation. The signaling pathways that are involved in the control of foam cell formation are not fully understood. In this study, we have investigated the role of phosphoinositide 3-kinase (PI3K) in relation to foam cell formation in human macrophages. The pan PI3K inhibitor LY294002 attenuated the uptake of modified LDL and macropinocytosis, as measured by Lucifer Yellow uptake, by human macrophages. In addition, the expression of SR-A, CD36 and LPL was attenuated by LY294002. The use of isoform-selective PI3K inhibitors showed that PI3K-β, -γ and -δ were all required for the expression of SR-A and CD36 whereas only PI3K-γ was necessary in the case of LPL. These studies reveal a pivotal role of PI3K in the control of macrophage foam cell formation and provide further evidence for their potential as therapeutic target against atherosclerosis. PMID:25663263

  17. Fucoidan inhibits the migration and proliferation of HT-29 human colon cancer cells via the phosphoinositide-3 kinase/Akt/mechanistic target of rapamycin pathways

    PubMed Central

    HAN, YONG-SEOK; LEE, JUN HEE; LEE, SANG HUN

    2015-01-01

    Fucoidan, a sulfated polysaccharide, has a variety of biological activities, including anti-cancer, anti-angiogenic and anti-inflammatory effects. However, the underlying mechanisms of fucoidan as an anti-cancer agent remain to be elucidated. The present study examined the anti-cancer effect of fucoidan on HT-29 human colon cancer cells. The cell growth of HT29 cells was significantly decreased following treatment with fucoidan (200 μg/ml). In addition, fucoidan inhibited the migration of HT-29 cells by decreasing the expression levels of matrix metalloproteinase-2 in a dose-dependent manner (0–200 μg/ml). The underlying mechanism of these inhibitory effects included the downregulation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) by treatment with fucoidan. Furthermore, fucoidan increased the expression of cleaved caspase-3 and decreased cancer sphere formation. The present study suggested that fucoidan exerts an anti-cancer effect on HT-29 human colon cancer cells by downregulating the PI3K-Akt-mTOR signaling pathway. Therefore, fucoidan may be a potential therapeutic reagent against the growth of human colon cancer cells. PMID:25998232

  18. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.

    PubMed

    Manning, Brendan D; Tee, Andrew R; Logsdon, M Nicole; Blenis, John; Cantley, Lewis C

    2002-07-01

    The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. We also show that S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and that T1462 is constitutively phosphorylated in PTEN(-/-) tumor-derived cell lines. Finally, we find that a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity. PMID:12150915

  19. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  20. RNA interference-mediated knockdown of Aurora-B alters the metastatic behavior of A549 cells via modulation of the phosphoinositide 3-kinase/Akt signaling pathway

    PubMed Central

    ZHOU, LONG DIAN; XIONG, XU; LONG, XIN HUA; LIU, ZHI LI; HUANG, SHAN HU; ZHANG, WEI

    2014-01-01

    Accumulating evidence has revealed that an elevated expression level of Aurora-B is associated with metastasis in various types of malignant tumor. However, it is currently unclear whether this molecule is involved in non-small lung cancer (NSCLC) metastasis, and the molecular mechanisms associated with Aurora-B and metastasis remain unknown. In the present study, in order to investigate whether Aurora-B is involved in the development and metastasis of NSCLC, the Aurora-B protein expression in NSCLC tissues was detected by immunohistochemistry and its association with metastasis was analyzed. The results revealed that the expression levels of the Aurora-B protein in tissues obtained from NSCLC patients with lymph node metastasis were significantly higher than those without metastatic disease. Furthermore, the effect of Aurora-B inhibition on A549 cell migration and invasion, as well as the activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was evaluated. Aurora-B was inhibited in the A549 cells using short hairpin RNA, and the cell migration and invasion rates were investigated using wound healing and Transwell invasion assays. In addition, the expression of the main proteins in the PI3K/Akt/nuclear factor-κB (NF-κB) signaling pathway, and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. The results demonstrated that cell migration and invasion were decreased as a result of silencing Aurora-B. Furthermore, the activity of the PI3K/Akt/NF-κB signaling pathway and the expression of MMP-2 and -9 protein were suppressed by silencing Aurora-B. The results of the present study indicate that the knockdown of Aurora-B suppresses A549 cell invasion and migration via the inhibition of the PI3K/Akt signaling pathway in vitro and thus, targeting Aurora-B may present a potential treatment strategy for NSCLC. PMID:25295091

  1. Inhibitory Effects of Isoquinoline Alkaloid Berberine on Ischemia-Induced Apoptosis via Activation of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway

    PubMed Central

    Kim, Mia; Shin, Mal Soon; Lee, Jae Min; Cho, Han Sam; Kim, Chang Ju; Kim, Young Joon; Choi, Hey Ran

    2014-01-01

    Purpose Berberine is a type of isoquinoline alkaloid that has been used to treat various diseases. A neuroprotective effect of berberine against cerebral ischemia has been reported; however, the effects of berberine on apoptosis in relation to reactive astrogliosis and microglia activation under ischemic conditions have not yet been fully evaluated. In the present study, we investigated the effects of berberine on global ischemia-induced apoptosis, and focused on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the hippocampus using gerbils. Methods Gerbils received berberine orally once a day for 14 consecutive days, starting one day after surgery. In this study, a step-down avoidance task was used to assess short-term memory. Furthermore, we employed the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to evaluate DNA fragmentation, immunohistochemistry to investigate glial fibriallary acidic protein, CD11b, and caspase-3, and western blot to assess PI3K, Akt, Bax, Bcl-2, and cytochrome c. Results Our results revealed that berberine treatment alleviated ischemia-induced short-term memory impairment. Treatment with berbeine also attenuated ischemia-induced apoptosis and inhibited reactive astrogliosis and microglia activation. Furthermore, berberine enhanced phospho-PI3K and phospho-Akt expression in the hippocampus of ischemic gerbils. Conclusions Berberine exerted a neuroprotective effect against ischemic insult by inhibiting neuronal apoptosis via activation of the PI3K/Akt signaling pathway. The antiapoptotic effect of berberine was achieved through inhibition of reactive astrogliosis and microglia activation. Berberine may therefore serve as a therapeutic agent for stroke-induced neurourological problems. PMID:25279238

  2. Insulin and insulin-like growth factor-1 can modulate the phosphoinositide-3-kinase/Akt/FoxO1 pathway in SZ95 sebocytes in vitro.

    PubMed

    Mirdamadi, Yasaman; Thielitz, Anja; Wiede, Antje; Goihl, Alexander; Papakonstantinou, Eleni; Hartig, Roland; Zouboulis, Christos C; Reinhold, Dirk; Simeoni, Luca; Bommhardt, Ursula; Quist, Sven; Gollnick, Harald

    2015-11-01

    A recent hypothesis suggests that a high glycaemic load diet-associated increase of insulin-like growth factor-1 (IGF-1) and insulin may promote acne by reducing nuclear localization of the forkhead box-O1 (FoxO1) transcription factor via activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. Using SZ95 sebocytes as a model, we investigated the effect of the most important insulinotropic western dietary factors, IGF-1 and insulin on acne. SZ95 sebocytes were stimulated with different concentrations of IGF-1 and insulin (0.001, 0.01, 0.1 and 1 μM) for 15 to 120 min ± PI3K inhibitor LY294002 (50 μM). Cytoplasmic and nuclear protein expression of p-Akt and p-FoxO1 as well as FoxO transcriptional activity was analysed. In addition, the proliferation and differentiation of sebocytes and their TLR2/4 expression were determined. We found that high concentrations of IGF-1 and insulin differentially stimulate the PI3K/Akt/FoxO1 pathway by an early up-regulation of cytoplasmic p-Akt and delayed up-regulation of p-FoxO1 resulting in FoxO1 shift to the cytoplasm and the reduction of FoxO transcriptional activity, physiological serum concentration had no effect. IGF-1 at concentrations of 0.1 and 1 μM significantly reduced proliferation but increased differentiation of sebocytes to a greater extent than insulin (0.1 and 1 μM), but up-regulated TLR2/4 expression to comparable extent. These data provide the first in vitro evidence that FoxO1 principally might be involved in the regulation of growth-factor-stimulatory effects on sebaceous lipogenesis and inflammation in the pathological condition of acne. However, the in vivo significance under physiological conditions remains to be elucidated. PMID:26257240

  3. Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

    PubMed

    Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

    2014-04-01

    Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. PMID:24168893

  4. Integrin β1-mediated acquired gefitinib resistance in non-small cell lung cancer cells occurs via the phosphoinositide 3-kinase-dependent pathway

    PubMed Central

    DENG, QIN-FANG; SU, BO; ZHAO, YIN-MIN; TANG, LIANG; ZHANG, JIE; ZHOU, CAI-CUN

    2016-01-01

    The present study aimed to explore the role of integrin β1 and the relevant signaling pathways in acquired gefitinib resistance in non-small cell lung cancer (NSCLC). The inhibitory effects of gefitinib, with or without LY294002, on cellular proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were analyzed by flow cytometry, while western blotting was used to evaluate the expression of EGFR, phosphorylated (phospho)-EGFR, protein kinase B (Akt), phospho-Akt, extracellular signal-regulated kinase (Erk) and phospho-Erk. The gene expression profiles of PC9 and PC9/G cells were determined by DNA microarray. Integrin β1 was knocked down in PC9/G cells by transiently transfected short interfering RNA (siRNA). A scrambled siRNA sequence was used as a control. Apoptosis of transfected cells was determined by Annexin V-phycoerythrin-Cy5/propidium iodide staining. Sequencing products were amplified by nested PCR. The resistant index of PC9/G cells to gefitinib was ~138- to 256-fold higher than that of PC9 cells, and this resistance was accompanied by significant increase in integrin β1 expression in PC9/G cells. Knockdown of integrin β1 with short hairpin RNA in PC9/G cells markedly inhibited proliferation and enhanced apoptosis in response to gefitinib, restoring the sensitivity of PC9/G cells gefitinib. Phosphoinositide 3-kinase (PI3K)/Akt activation was observed in PC9/G cells in the presence of gefitinib and the sensitivity of PC9/G cells to gefitinib was also able to be restored by PI3K/Akt pathway inhibitor LY294002. Finally, knockdown of integrin β1 significantly reduced the levels of phospho-Akt. These findings suggest that integrin β1 signaling via the PI3K/Akt pathway may be a significant mechanism underlying gefitinib resistance, and may potentially present an alternative therapeutic target for the treatment of NSCLC unresponsive to EGFR inhibitors. PMID:26870244

  5. Phosphoinositide 3-kinase enhancer (PIKE) in the brain: is it simply a phosphoinositide 3-kinase/Akt enhancer?

    PubMed Central

    Chan, Chi Bun; Ye, Keqiang

    2013-01-01

    Since its discovery in 2000, phosphoinositide 3-kinase enhancer (PIKE) has been recognized as a class of GTPase that controls the enzymatic activities of phosphoinositide 3-kinase (PI3K) and Akt in the central nervous system (CNS). However, recent studies suggest that PIKEs are not only enhancers to PI3K/Akt but also modulators to other kinases including insulin receptor tyrosine kinase and focal adhesion kinases. Moreover, they regulate transcription factors such as signal transducer and activator of transcription and nuclear factor κB. Indeed, PIKE proteins participate in multiple cellular processes including control of cell survival, brain development, memory formation, gene transcription, and metabolism. In this review, we have summarized the functions of PIKE proteins in CNS and discussed their potential implications in various neurological disorders. PMID:22499674

  6. Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway.

    PubMed

    Myou, Shigeharu; Leff, Alan R; Myo, Saori; Boetticher, Evan; Meliton, Angelo Y; Lambertino, Anissa T; Liu, Jie; Xu, Chang; Munoz, Nilda M; Zhu, Xiangdong

    2003-10-15

    Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK. PMID:14530366

  7. Interaction of Ras with phosphoinositide 3-kinase gamma.

    PubMed Central

    Rubio, I; Rodriguez-Viciana, P; Downward, J; Wetzker, R

    1997-01-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) can be activated in vitro by both alpha and betagamma subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kalpha. Here we demonstrate the binding of Ras to PI3Kgamma in vitro. An N-terminal region of PI3Kgamma was identified as a binding site for Ras. After co-expression with PI3Kgamma in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kalpha by Ras in the same cells. PMID:9307042

  8. A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

    PubMed Central

    Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

    2011-01-01

    Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates

  9. HspB8 mediates neuroprotection against OGD/R in N2A cells through the phosphoinositide 3-kinase/Akt pathway.

    PubMed

    Hu, Zhiping; Yang, Binbin; Mo, Xiaoye; Zhou, Fangfang

    2016-08-01

    In a previous study, we found that Heat shock protein B8 (HspB8) overexpression could prevent the apoptosis and reduced cell viability induced by OGD/R and showed that the neuroprotective effect of HspB8 was mediated by inhibition of the mitochondrial apoptotic pathway. In recent study, HspB8 has been shown to protect the heart against ischemia/reperfusion (I/R) injury via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, whether this protective effect applied to brain I/R injury remained unexplored. To further test the mechanism of HspB8's effects in brain, we used oxygen-glucose deprivation followed by reperfusion (OGD/R), an in vitro model of ischemia to examine the involvement of PI3K/Akt signaling by treating mouse neuroblastoma cells (N2A cells) (untransfected or transfected with an HspB8 expression vector) with the PI3K inhibitor LY294002 before OGD/R. Our results revealed that the apoptosis-suppressing effect of HspB8 was mediated by the PI3K/Akt pathway. Therefore, HspB8 protected the N2A cells against OGD/R insult, possibly by activating the PI3K/Akt signaling pathway. PMID:27178361

  10. Deletion of the phosphoinositide 3-Kinase p110(gamma) gene attenuates murine atherosclerosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, ch...

  11. Inhibition of aldose reductase prevents growth factor-induced G1-S phase transition through the AKT/phosphoinositide 3-kinase/E2F-1 pathway in human colon cancer cells.

    PubMed

    Ramana, Kota V; Tammali, Ravinder; Srivastava, Satish K

    2010-04-01

    Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR. PMID:20354121

  12. Protection against 1-methyl-4-phenyl pyridinium-induced neurotoxicity in human neuroblastoma SH-SY5Y cells by Soyasaponin I by the activation of the phosphoinositide 3-kinase/AKT/GSK3β pathway.

    PubMed

    Guo, Zheng; Cao, Wei; Zhao, Shifeng; Han, Zengtai; Han, Boxiang

    2016-07-01

    Parkinson's disease (PD) can be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of Soyasaponin I (Soya-I), a naturally occurring triterpene extracted from a widely used ingredient in many foods, such as Glycine max (soybean), were evaluated in a widely used cellular PD model in which neurotoxicity was induced by 1-methyl-4-phenyl pyridinium (MPP) in cultured SH-SY5Y cells. We found that Soya-I at 10-40 μM considerably protected against MPP-induced neurotoxicity as evidenced by an increase in cell viability, a decrease in lactate dehydrogenase release, and a reduction in apoptotic nuclei. Moreover, Soya-I effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as the Bax/Bcl-2 ratio caused by MPP. Most importantly, Soya-I markedly reversed the inhibition of protein expression of phosphorylated AKT and phosphorylated GSK3β caused by MPP. LY294002, the specific inhibitor of phosphoinositide 3-kinase, significantly abrogated the upregulated phosphorylated AKT and phosphorylated GSK3β offered by Soya-I, suggesting that the neuroprotection of Soya-I was mainly dependent on the activation of the phosphoinositide 3-kinase/AKT/GSK3β signaling pathway. The results taken together indicate that Soya-I may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD. PMID:27196724

  13. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  14. Classes of phosphoinositide 3-kinases at a glance

    PubMed Central

    Jean, Steve; Kiger, Amy A.

    2014-01-01

    ABSTRACT The phosphoinositide 3-kinase (PI3K) family is important to nearly all aspects of cell and tissue biology and central to human cancer, diabetes and aging. PI3Ks are spatially regulated and multifunctional, and together, act at nearly all membranes in the cell to regulate a wide range of signaling, membrane trafficking and metabolic processes. There is a broadening recognition of the importance of distinct roles for each of the three different PI3K classes (I, II and III), as well as for the different isoforms within each class. Ongoing issues include the need for a better understanding of the in vivo complexity of PI3K regulation and cellular functions. This Cell Science at a Glance article and the accompanying poster summarize the biochemical activities, cellular roles and functional requirements for the three classes of PI3Ks. In doing so, we aim to provide an overview of the parallels, the key differences and crucial interplays between the regulation and roles of the three PI3K classes. PMID:24587488

  15. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation

    PubMed Central

    Pritchard, Rory A.; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S.

    2016-01-01

    Abstract Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  16. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  17. Implication of the calcium sensing receptor and the Phosphoinositide 3-kinase/Akt pathway in the extracellular calcium-mediated migration of RAW 264.7 osteoclast precursor cells.

    PubMed

    Boudot, Cédric; Saidak, Zuzana; Boulanouar, Abdel Krim; Petit, Laurent; Gouilleux, Fabrice; Massy, Ziad; Brazier, Michel; Mentaverri, Romuald; Kamel, Saïd

    2010-05-01

    While the processes involved in the formation, maturation and apoptosis of osteoclasts have been investigated extensively in previous studies, little is known about the mechanisms responsible for the localization and homing of osteoclast precursor cells to the bone environment in order to initiate the bone remodeling process. Recent studies have suggested that the extracellular Ca(2+) (Ca(2+)(o)) concentration gradient present near the bone environment may be one of the participating factors, producing a chemoattractant effect on osteoclast precursors. Using the murine osteoclast precursor cells of the monocyte-macrophage lineage, the RAW 264.7 cell line, we have shown that Ca(2+)(o) increases the migration of these cells in a directional manner. The participation of the calcium sensing receptor (CaR) in this effect was tested by knocking down its expression through RNA interference, which resulted in an abolition of the migratory response. By the use of specific pathway inhibitors and western blot analysis, the phosphoinositide 3-kinase (PI3K)/Akt and phospholipase Cbeta pathways were shown to be implicated in the migratory effect. The implication of the Akt pathway in the Ca(2+)(o)-induced chemoattraction of RAW 264.7 cells was also confirmed by transducing the cells with the fusion protein TAT-dominant negative-Akt, which decreased the migratory effect. In contrast, the MAPK pathways (ERK1/2, p38 and JNK) were not involved in the production of the migratory effect. We conclude that through the activation of the CaR and subsequent signaling via the PI3K/Akt pathway, Ca(2+)(o) produces a chemoattractant effect on the osteoclast precursor RAW 264.7 cells. These results suggest that the Ca(2+)(o) gradient present near the bone may be one of the initiating factors for the homing of osteoclast precursors to bone, thus possibly playing a role in the initiation of bone remodeling. PMID:20149906

  18. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways

    PubMed Central

    CHUANG, WAN-LING; SU, CHIN-CHENG; LIN, PING-YI; LIN, CHI-CHEN; CHEN, YAO-LI

    2015-01-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. PMID:25847489

  19. Berberine Induced Apoptosis of Human Osteosarcoma Cells by Inhibiting Phosphoinositide 3 Kinase/Protein Kinase B (PI3K/Akt) Signal Pathway Activation

    PubMed Central

    2016-01-01

    Background: Osteosarcoma is a malignant tumor with high mortality but effective therapy has not yet been developed. Berberine, an isoquinoline alkaloid component in several Chinese herbs including Huanglian, has been shown to induce growth inhibition and the apoptosis of certain cancer cells. The aim of this study was to determine the role of berberine on human osteosarcoma cell lines U2OS and its potential mechanism. Methods: The proliferation effect of U20S was exanimed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and the percentage of apoptotic cells were determined by flow cytometric analysis. The expression of PI3K, p-Akt, Bax, Bcl-2, cleavage-PARP and Caspase3 were detected by Western blott. Results: Berberine treatment caused dose-dependent inhibiting proliferation and inducing apoptosis of U20S cell. Mechanistically, berberine inhibits PI3K/AKT activation that, in turn, results in up-regulating the expression of Bax, and PARP and down-regulating the expression of Bcl-2 and caspase3. In all, berberine can suppress the proliferation and induce the apoptosis of U2OS cell through inhibiting the PI3K/Akt signaling pathway activation. Conclusion: Berberine can suppress the proliferation and induce the apoptosis of U2OS cell through inhibiting the PI3K/Akt signaling pathway activation. PMID:27398330

  20. (Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

    PubMed Central

    Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

    2011-01-01

    In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

  1. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase.

    PubMed Central

    Conway, A M; Rakhit, S; Pyne, S; Pyne, N J

    1999-01-01

    The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade. PMID:9882612

  2. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase.

    PubMed Central

    Rakhit, S; Conway, A M; Tate, R; Bower, T; Pyne, N J; Pyne, S

    1999-01-01

    We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis. PMID:10051434

  3. Roles of MKK1/2-ERK1/2 and phosphoinositide 3-kinase-AKT signaling pathways in erlotinib-induced Rad51 suppression and cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Ciou, Shih-Ci; Jhan, Jhih-Yuan; Cheng, Chao-Min; Su, Ying-Jhen; Chuang, Show-Mei; Lin, Szu-Ting; Chang, Chia-Che; Lin, Yun-Wei

    2009-08-01

    Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC. PMID:19671683

  4. The phosphoinositide 3-kinase/Akt-signal pathway mediates proliferation and secretory function of hepatic sinusoidal endothelial cells in rats after partial hepatectomy

    SciTech Connect

    Chen Ping . E-mail: chenping@263.net; Zhang Lin; Ding Jiming; Zhu Jin; Li Ying; Duan Shigang; Yan Hongtao; Huan Yongwei; Dong Jiahong

    2006-04-14

    Objective: To investigate the role of AKT signaling pathway in hepatic sinusoidal endothelial cells (SECs) early after partial hepatectomy in rats and the regulatory mechanisms involved. Methods: The animal model of 70% hepatectomy was made. Hepatic SECs were isolated and cultured according to Braet et al.'s method with some modifications. The cultured hepatic SECs were divided into two groups: 70% partial hepatectomy groups and LY294002 group (LY). We observed the expressions of AKT and NF-{kappa}B in cultured hepatic SECs by Western blot, measured the levels of NO, NOs, IL-6, and HGF in the supernatants of hepatic SEC cultures and [{sup 3}H]thymidine incorporation, and analyzed cell cycle of cultured hepatic SECs by flow cytometer. The relationship of the Akt pathway with secretions and proliferation of hepatic SECs after partial hepatectomy was probed. Results: The levels of Akt protein expression increased significantly after partial hepatectomy in OG group and with a peak at 24 h post operation. Meanwhile, there was a markedly increase in phosphorylated Akt protein during 2-72 h after operation. But the expression and activity of Akt protein did not change significantly after partial hepatectomy in the LY group. So, partial hepatectomy can marked induce Akt expression and result in rapid and marked phosphorylation of Akt from 2 to 72 h thereafter. The changes of NF-{kappa}B expression in cultured hepatic SECs were similar to those of Akt expression after operation. The concentrations of HGF and IL-6 in the supernatants of cultured hepatic SECs were relatively low in the LY group, and were markedly increased after partial hepatectomy, with a peak at 24 h in the OG group. There were significant differences between the OG and LY groups at 6 and 24 h (P < 0.05). Both NO and NOS secretion was increased in the OG group compared to the LY group within 24 h after partial hepatectomy. But the secretion of NO and NOS was increased more markedly in the LY group than that

  5. Phosphoinositide 3-kinase (PI3K) and the nutrient sensing mTOR (mammalian target of rapamycin) pathways control T cell migration

    PubMed Central

    Finlay, David; Cantrell, Doreen

    2012-01-01

    The established role for Phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3) signalling pathways is to regulate cell metabolism. More recently it has emerged that PI(3,4,5)P3 signalling via mTOR and Foxo transcription factors also controls lymphocyte trafficking by determining the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. In quiescent T cells, non-phosphorylated active Foxos maintain expression of KLF2, a transcription factor that regulates expression of the chemokine receptors CCR7 and S1P1and the adhesion receptor CD62L that together control T cell transmigration into secondary lymphoid tissues. PI(3,4,5)P3 mediated activation of Protein Kinase B phosphorylates and inactivates Foxos thereby terminating expression of KLF2 and its target genes. The correct localization of lymphocytes is essential for effective immune responses and the ability of PI3K and mTOR to regulate expression of chemokine receptor and adhesion molecules puts these signaling molecules at the core of the molecular mechanisms that control lymphocyte trafficking. PMID:20146713

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

    PubMed

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

    2014-07-18

    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 Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 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 Li2CO3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li2CO3 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 Li2CO3 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. PMID:24950409

  7. Neuregulin-1 Regulates Cell Adhesion via an ErbB2/Phosphoinositide-3 Kinase/Akt-Dependent Pathway: Potential Implications for Schizophrenia and Cancer

    PubMed Central

    Kanakry, Christopher G.; Li, Zhen; Nakai, Yoko; Sei, Yoshitatsu; Weinberger, Daniel R.

    2007-01-01

    Background Neuregulin-1 (NRG1) is a putative schizophrenia susceptibility gene involved extensively in central nervous system development as well as cancer invasion and metastasis. Using a B lymphoblast cell model, we previously demonstrated impairment in NRG1α-mediated migration in cells derived from patients with schizophrenia as well as effects of risk alleles in NRG1 and catechol-O-methyltransferase (COMT), a second gene implicated both in schizophrenia susceptibility and in cancer. Methodology/Principal Findings Here, we examine cell adhesion, an essential component process of cell motility, using an integrin-mediated cell adhesion assay based on an interaction between ICAM-1 and the CD11a/CD18 integrin heterodimer expressed on lymphoblasts. In our assay, NRG1α induces lymphoblasts to assume varying levels of adhesion characterized by time-dependent fluctuations in the firmness of attachment. The maximum range of variation in adhesion over sixty minutes correlates strongly with NRG1α-induced migration (r2 = 0.61). NRG1α-induced adhesion variation is blocked by erbB2, PI3K, and Akt inhibitors, but not by PLC, ROCK, MLCK, or MEK inhibitors, implicating the erbB2/PI3K/Akt1 signaling pathway in NRG1-stimulated, integrin-mediated cell adhesion. In cell lines from 20 patients with schizophrenia and 20 normal controls, cells from patients show a significant deficiency in the range of NRG1α-induced adhesion (p = 0.0002). In contrast, the response of patient-derived cells to phorbol myristate acetate is unimpaired. The COMT Val108/158Met genotype demonstrates a strong trend towards predicting the range of the NRG1α-induced adhesion response with risk homozygotes having decreased variation in cell adhesion even in normal subjects (p = 0.063). Conclusion/Significance Our findings suggest that a mechanism of the NRG1 genetic association with schizophrenia may involve the molecular biology of cell adhesion. PMID:18159252

  8. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

    PubMed Central

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-01-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases. PMID:26018563

  9. Exendin-4 enhances the migration of adipose-derived stem cells to neonatal rat ventricular cardiomyocyte-derived conditioned medium via the phosphoinositide 3-kinase/Akt-stromal cell-derived factor-1α/CXC chemokine receptor 4 pathway

    PubMed Central

    ZHOU, HAO; YANG, JUNJIE; XIN, TING; ZHANG, TAO; HU, SHUNYIN; ZHOU, SHANSHAN; CHEN, GUANGHUI; CHEN, YUNDAI

    2015-01-01

    Adipose-derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex-4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte-derived conditioned medium (NRVC-CM). The ADSCs and cardiomyocytes were cultured in vitro. Following incubation of the ADSCs with Ex-4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT-qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell-derived factor-1α (SDF-1α) were evaluated in the NRVC-CM treated with Ex-4 by ELISA, RT-qPCR and western blot analysis. The migration of the ADSCs to the NRVC-CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p−)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex-4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF-1α in the cardiomyocytes and increased the expression levels of p-Akt in both cells. However, the alterations to the SDF-1α/C XC R4 cascade in the cells were abrogated following pretreatment with LY-294002, a phosphoinositide 3-kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC-CM, following treatment with Ex-4. However, these effects were reduced significantly by pretreatment of the cells with the SDF-1

  10. Control of Cardiac Repolarization by Phosphoinositide 3-kinase Signaling to Ion Channels

    PubMed Central

    Ballou, Lisa M.; Lin, Richard Z.; Cohen, Ira S.

    2014-01-01

    Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long QT syndrome by direct block of the rapid delayed rectifier (IKr) also appear to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes also may result from changes in multiple currents due to decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the impact of PI3K and its downstream effectors including Akt on sodium, potassium and calcium currents in cardiac myocytes. We also refer to some studies in non-cardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility. PMID:25552692

  11. PIKE GTPase are phosphoinositide-3-kinase enhancers, suppressing programmed cell deathPIKE GTPase are phosphoinositide-3-kinase enhancers, suppressing programmed cell death

    PubMed Central

    Chan, Chi Bun; Ye, Keqiang; Chan, Chi Bun; Ye, Keqiang

    2007-01-01

    Abstract Phosphoinositide-3-kinase enhancers (PIKE) are GTP-binding proteins that posses anti-apoptotic functions. The PIKE family includes three members, PIKE-L, PIKE-S and PIKE-A, which are originated from a single gene (CENTG1) through alternative splicing or differential transcription initiation. Both PIKE-S and PIKE-L bind to phosphoinositide-3-kinase (PI3K) and enhance its activity. PIKE-A does not interplay with PI3K. Instead, it interacts with the downstream effector Akt and promotes its activity. These actions are mediated by their GTPase activity. Because both PI3K and Akt are important effectors in the growth factor-mediated signaling which triggers cellular growth and acts against apoptosis, PIKEs therefore serve as the molecular switch that their activation are crucial for growth factors to exert their physiological functions. In this review, the current understanding of different PIKE isoforms in growth factors-induced anti-apoptotic function will be discussed. Moreover, the role of PIKE in the survival and invasion activity of cancer cells will also be introduced. PMID:17367500

  12. Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

    PubMed Central

    Fruman, David A.; Ferl, Gregory Z.; An, Sam S.; Donahue, Amber C.; Satterthwaite, Anne B.; Witte, Owen N.

    2002-01-01

    Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle. PMID:11756681

  13. Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

    SciTech Connect

    Ng, Stanley K.L.; Neo, Soek-Ying; Yap, Yann-Wan; Karuturi, R. Krishna Murthy; Loh, Evelyn S.L.; Liau, Kui-Hin; Ren, Ee-Chee

    2009-09-18

    Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

  14. 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.

  15. The intricate regulation and complex functions of the Class III phosphoinositide 3-kinase Vps34.

    PubMed

    Backer, Jonathan M

    2016-08-01

    The Class III phosphoinositide 3-kinase Vps34 (vacuolar protein sorting 34) plays important roles in endocytic trafficking, macroautophagy, phagocytosis, cytokinesis and nutrient sensing. Recent studies have provided exciting new insights into the structure and regulation of this lipid kinase, and new cellular functions for Vps34 have emerged. This review critically examines the wealth of new data on this important enzyme, and attempts to integrate these findings with current models of Vps34 signalling. PMID:27470591

  16. Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

    PubMed Central

    Hehl, S; Stoyanov, B; Oehrl, W; Schönherr, R; Wetzker, R; Heinemann, S H

    2001-01-01

    Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect. PMID:11736661

  17. Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

    PubMed Central

    Cummings, Hannah E.; Barbi, Joseph; Reville, Patrick; Oghumu, Steve; Zorko, Nicholas; Sarkar, Anasuya; Keiser, Tracy L.; Lu, Bao; Rückle, Thomas; Varikuti, Sanjay; Lezama-Davila, Claudio; Wewers, Mark D.; Whitacre, Caroline; Radzioch, Danuta; Rommel, Christian; Seveau, Stéphanie; Satoskar, Abhay R.

    2012-01-01

    Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania. PMID:22232690

  18. The p110α Isoform of Phosphoinositide 3-Kinase is Essential for Cone Photoreceptor Survival

    PubMed Central

    Rajala, Raju V.S.; Ranjo-Bishop, Michelle; Wang, Yuhong; Rajala, Ammaji; Anderson, Robert E.

    2015-01-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions. PMID:25742742

  19. Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase

    PubMed Central

    2013-01-01

    Background Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. Methods A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. Results Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. Conclusions Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production. PMID:23680019

  20. Phosphoinositide-3-Kinase Is the Primary Mediator of Phosphoinositide-Dependent Inhibition in Mammalian Olfactory Receptor Neurons

    PubMed Central

    Ukhanov, Kirill; Corey, Elizabeth; Ache, Barry W.

    2016-01-01

    Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs. PMID:27147969

  1. Phosphoinositide 3-kinase dependent regulation of Kv channels in dendritic cells.

    PubMed

    Shumilina, Ekaterina; Zahir, Naima; Xuan, Nguyen Thi; Lang, Florian

    2007-01-01

    The phosphoinositide 3 (PI3) kinase plays a pivotal role in the regulation of dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory. PI3 kinase is an endogenous suppressor of interleukin 12 (IL-12) production in DCs that is triggered by Toll-like receptor signaling. Inhibition of IL-12 production limits T helper 1 (Th1) polarization. On the other hand, PI3 kinase is an important regulator of various ion channels. The present study aimed to explore whether ion channels in DCs are regulated by PI3 kinase and whether they are important for DC function. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by patch clamp. As a result, DCs express voltage-gated K(+) channels (Kv), which are blocked by Stichodactyla helianthus toxin (ShK, 2.5 nM). A significant upregulation of Kv currents was observed upon maturation of DCs as induced by stimulation of the cells with lipopolysaccharide (LPS, 0.1 microg/ml, 48 h). A dramatic increase of Kv current amplitude was observed following preincubation of the cells with LY294002 (100 nM), a specific inhibitor of PI3 kinase. PI3 kinase inhibitor wortmannin (100 nM) similarly increased Kv current. LY294002 treatment was further followed by a significant increase of IL-12 production. ShK (100 nM) significantly blunted the stimulation of IL-12 release by LPS but not when the cells were first pretreated with LY294002. The observations point to Kv channel sensitive and Kv channel insensitive regulation of DC function. PMID:17982262

  2. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  3. Fibroblast Growth Factor Receptor-2 Contributes to the Basic Fibroblast Growth Factor-Induced Neuronal Differentiation in Canine Bone Marrow Stromal Cells via Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Nakano, Rei; Edamura, Kazuya; Nakayama, Tomohiro; Narita, Takanori; Okabayashi, Ken; Sugiya, Hiroshi

    2015-01-01

    Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs. PMID:26523832

  4. An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo

    PubMed Central

    De, Pradip; Dey, Nandini; Terakedis, Breanne; Bersagel, Leif; Li, Zhi Hua; Mahadevan, Daruka; Garlich, Joseph R.; Trudel, Suzanne; Makale, Milan T.; Durden, Donald L.

    2013-01-01

    Purpose Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including multiple myeloma, and (2) to begin combining pan PI-3 kinase inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. Methods We determined the in vitro activity of SF1126 with16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. Results SF1126 was cytotoxic to all tested MM lines and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1α. Tumor growth was 94% inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient derived MM tumor cells in vivo. Conclusions Our results establish three important points: (1) SF1126, a pan PI-3 kinase inhibitor has potent antitumor activity against multiple myeloma in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade®, and (3) SF1126 blocks the IGF-1 induced activation of AKT in primary MM tumor cells isolated from SF1126 treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies. PMID:23355037

  5. Phosphoinositide 3-kinase gamma (PI3Kgamma) inhibitors for the treatment of inflammation and autoimmune disease.

    PubMed

    Venable, Jennifer D; Ameriks, Michael K; Blevitt, Jonathan M; Thurmond, Robin L; Fung-Leung, Wai-Ping

    2010-01-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) is a lipid kinase in leukocytes that generates phosphatidylinositol 3,4,5-trisphosphate to recruit and activate downstream signaling molecules. Distinct from other members in the PI3K family, PI3Kgamma is activated by G-protein coupled-receptors responding to chemotactic ligands. PI3Kgamma plays an important role in migration of both myeloid and lymphoid cells. It is also required for other leukocyte functions such as neutrophil oxidative burst, T cell proliferation and mast degranulation. Mice with PI3Kgamma inactivated by genetic or pharmacological approaches are protected from disease development in a number of inflammation and autoimmune disease models. The function of PI3Kgamma depends on its kinase activity and therefore it has been suggested by many reports that small molecules inhibiting its kinase activity could be promising for the treatment of inflammation and autoimmune diseases. Over the last five years, a number of pharmaceutical companies have reported a wide variety of PI3Kgamma inhibitors, of which several x-ray crystal structures with PI3Kgamma have been elucidated. The structural characteristics and selectivity profiles of these inhibitors, in particular thiazolidinones and 2-aminoheterocycles, and those disclosed in related patent applications are summarized in this review. PMID:20017720

  6. Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk

    PubMed Central

    Stevens, K N; Garcia-Closas, M; Fredericksen, Z; Kosel, M; Pankratz, V S; Hopper, J L; Dite, G S; Apicella, C; Southey, M C; Schmidt, M K; Broeks, A; Van ‘t Veer, L J; Tollenaar, R A E M; Fasching, P A; Beckmann, M W; Hein, A; Ekici, A B; Johnson, N; Peto, J; dos Santos Silva, I; Gibson, L; Sawyer, E; Tomlinson, I; Kerin, M J; Chanock, S; Lissowska, J; Hunter, D J; Hoover, R N; Thomas, G D; Milne, R L; Pérez, JI Arias; González-Neira, A; Benítez, J; Burwinkel, B; Meindl, A; Schmutzler, R K; Bartrar, C R; Hamann, U; Ko, Y D; Brüning, T; Chang-Claude, J; Hein, R; Wang-Gohrke, S; Dörk, T; Schürmann, P; Bremer, M; Hillemanns, P; Bogdanova, N; Zalutsky, J V; Rogov, Y I; Antonenkova, N; Lindblom, A; Margolin, S; Mannermaa, A; Kataja, V; Kosma, V-M; Hartikainen, J; Chenevix-Trench, G; Chen, X; Peterlongo, P; Bonanni, B; Bernard, L; Manoukian, S; Wang, X; Cerhan, J; Vachon, C M; Olson, J; Giles, G G; Baglietto, L; McLean, C A; Severi, G; John, E M; Miron, A; Winqvist, R; Pylkäs, K; Jukkola-Vuorinen, A; Grip, M; Andrulis, I; Knight, J A; Glendon, G; Mulligan, A M; Cox, A; Brock, I W; Elliott, G; Cross, S S; Pharoah, P P; Dunning, A M; Pooley, K A; Humphreys, M K; Wang, J; Kang, D; Yoo, K-Y; Noh, D-Y; Sangrajrang, S; Gabrieau, V; Brennan, P; McKay, J; Anton-Culver, H; Ziogas, A; Couch, F J; Easton, D F

    2011-01-01

    Background: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. Methods: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). Results: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, P=4.6 × 10−3), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96–1.01, P=0.139). Conclusion: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer PMID:22033276

  7. Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

    PubMed

    Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L; Bowman, Amber L; Mangmool, Supachoke; Xiao, Kunhong; Naga Prasad, Sathyamangla V; Rockman, Howard A

    2016-10-01

    β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling. PMID:27169346

  8. The class I phosphoinositide 3-kinases α and β control antiphospholipid antibodies-induced platelet activation.

    PubMed

    Terrisse, Anne-Dominique; Laurent, Pierre-Alexandre; Garcia, Cédric; Gratacap, Marie-Pierre; Vanhaesebroeck, Bart; Sié, Pierre; Payrastre, Bernard

    2016-06-01

    Antiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies (aPL) associated with increased thrombotic risk and pregnancy morbidity. Although aPL are heterogeneous auto-antibodies, the major pathogenic target is the plasma protein β2-glycoprotein 1. The molecular mechanisms of platelet activation by aPL remain poorly understood. Here, we explored the role of the class IA phosphoinositide 3-kinase (PI3K) α and β isoforms in platelet activation by aPL. Compared to control IgG from healthy individuals, the IgG fraction isolated from patients with APS potentiates platelet aggregation induced by low dose of thrombin in vitro and increases platelet adhesion and thrombus growth on a collagen matrix under arterial shear rate through a mechanism involving glycoprotein Ib (GPIb) and Toll Like Receptor 2 (TLR-2). Using isoforms-selective pharmacological PI3K inhibitors and mice with megakaryocyte/platelet lineage-specific inactivation of class IA PI3K isoforms, we demonstrate a critical role of the PI3Kβ and PI3Kα isoforms in platelet activation induced by aPL. Our data show that aPL potentiate platelet activation through GPIbα and TLR-2 via a mechanism involving the class IA PI3Kα and β isoforms, which represent new potential therapeutic targets in the prevention or treatment of thrombotic events in patients with APS. PMID:26818901

  9. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    PubMed

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  10. Phosphoinositide 3-kinase γ/δ inhibition limits infarct size after myocardial ischemia/reperfusion injury

    PubMed Central

    Doukas, John; Wrasidlo, Wolfgang; Noronha, Glenn; Dneprovskaia, Elena; Fine, Richard; Weis, Sara; Hood, John; DeMaria, Anthony; Soll, Richard; Cheresh, David

    2006-01-01

    Although phosphoinositide 3-kinases (PI3Ks) play beneficial pro-cell survival roles during tissue ischemia, some isoforms (γ and δ) paradoxically contribute to the inflammation that damages these same tissues upon reperfusion. We therefore considered the possibility that selectively inhibiting proinflammatory PI3K isoforms during the reperfusion phase could ultimately limit overall tissue damage seen in ischemia/reperfusion injuries such as myocardial infarction. Panreactive and isoform-restricted PI3K inhibitors were identified by screening a novel chemical family; molecular modeling studies attributed isoform specificity based on rotational freedom of substituent groups. One compound (TG100-115) identified as a selective PI3K γ/δ inhibitor potently inhibited edema and inflammation in response to multiple mediators known to participate in myocardial infarction, including vascular endothelial growth factor and platelet-activating factor; by contrast, endothelial cell mitogenesis, a repair process important to tissue survival after ischemic damage, was not disrupted. In rigorous animal MI models, TG100-115 provided potent cardioprotection, reducing infarct development and preserving myocardial function. Importantly, this was achieved when dosing well after myocardial reperfusion (up to 3 h after), the same time period when patients are most accessible for therapeutic intervention. In conclusion, by targeting pathologic events occurring relatively late in myocardial damage, we have identified a potential means of addressing an elusive clinical goal: meaningful cardioprotection in the postreperfusion time period. PMID:17172449

  11. Phosphoinositide 3-kinase gamma/delta inhibition limits infarct size after myocardial ischemia/reperfusion injury.

    PubMed

    Doukas, John; Wrasidlo, Wolfgang; Noronha, Glenn; Dneprovskaia, Elena; Fine, Richard; Weis, Sara; Hood, John; Demaria, Anthony; Soll, Richard; Cheresh, David

    2006-12-26

    Although phosphoinositide 3-kinases (PI3Ks) play beneficial pro-cell survival roles during tissue ischemia, some isoforms (gamma and delta) paradoxically contribute to the inflammation that damages these same tissues upon reperfusion. We therefore considered the possibility that selectively inhibiting proinflammatory PI3K isoforms during the reperfusion phase could ultimately limit overall tissue damage seen in ischemia/reperfusion injuries such as myocardial infarction. Panreactive and isoform-restricted PI3K inhibitors were identified by screening a novel chemical family; molecular modeling studies attributed isoform specificity based on rotational freedom of substituent groups. One compound (TG100-115) identified as a selective PI3K gamma/delta inhibitor potently inhibited edema and inflammation in response to multiple mediators known to participate in myocardial infarction, including vascular endothelial growth factor and platelet-activating factor; by contrast, endothelial cell mitogenesis, a repair process important to tissue survival after ischemic damage, was not disrupted. In rigorous animal MI models, TG100-115 provided potent cardioprotection, reducing infarct development and preserving myocardial function. Importantly, this was achieved when dosing well after myocardial reperfusion (up to 3 h after), the same time period when patients are most accessible for therapeutic intervention. In conclusion, by targeting pathologic events occurring relatively late in myocardial damage, we have identified a potential means of addressing an elusive clinical goal: meaningful cardioprotection in the postreperfusion time period. PMID:17172449

  12. Short-Form Ron Promotes Spontaneous Breast Cancer Metastasis through Interaction with Phosphoinositide 3-Kinase

    PubMed Central

    Liu, Xuemei; Zhao, Ling; DeRose, Yoko S.; Lin, Yi-Chun; Bieniasz, Magdalena; Eyob, Henok; Buys, Saundra S.; Neumayer, Leigh

    2011-01-01

    Receptor tyrosine kinases (RTKs) have been the subject of intense investigation due to their widespread deregulation in cancer and the prospect of developing targeted therapeutics against these proteins. The Ron RTK has been implicated in tumor aggressiveness and is a developing target for therapy, but its function in tumor progression and metastasis is not fully understood. We examined Ron activity in human breast cancers and found striking predominance of an activated Ron isoform known as short-form Ron (sfRon), whose function in breast tumors has not been explored. We found that sfRon plays a significant role in aggressiveness of breast cancer in vitro and in vivo. sfRon expression was sufficient to convert slow-growing, nonmetastatic tumors into rapidly growing tumors that spontaneously metastasized to liver and bones. Mechanistic studies revealed that sfRon promotes epithelial-mesenchymal transition, invasion, tumor growth, and metastasis through interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K activity, or introduction of a single mutation in the p85 docking site on sfRon, completely eliminated the ability of sfRon to promote tumor growth, invasion, and metastasis. These findings reveal sfRon as an important new player in breast cancer and validate Ron and PI3K as therapeutic targets in this disease. PMID:22207901

  13. Dose-Dependent Suppression of Cytokine production from T cells by a Novel Phosphoinositide 3-Kinase Delta Inhibitor

    PubMed Central

    Way, Emily E.; Trevejo-Nunez, Giraldina; Kane, Lawrence P.; Steiner, Bart H.; Puri, Kamal D.; Kolls, Jay K.; Chen, Kong

    2016-01-01

    There remains a significant need for development of effective small molecules that can inhibit cytokine-mediated inflammation. Phosphoinositide 3 kinase (PI3K) is a direct upstream activator of AKT, and plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and PI3K inhibitors have been approved or are in clinical development. We examined novel PI3Kdelta inhibitors, which are highly selective for the p110delta isoform of in CD3/CD28 stimulated T-cell cytokine production. In vitro generated CD4+ T effector cells stimulated in the presence of a PI3Kdelta inhibitor demonstrated a dose-dependent suppression of cytokines produced by Th1, Th2, and Th17 cells. This effect was T-cell intrinsic, and we observed similar effects on human PBMCs. Th17 cells expressing a constitutively activated form of AKT were resistant to PI3Kdelta inhibition, suggesting that the inhibitor is acting through AKT signaling pathways. Additionally, PI3Kdelta inhibition decreased IL-17 production in vivo and decreased neutrophil recruitment to the lung in a murine model of acute pulmonary inflammation. These experiments show that targeting PI3Kdelta activity can modulate T-cell cytokine production and reduce inflammation in vivo, suggesting that PI3Kdelta inhibition could have therapeutic potential in treating inflammatory diseases. PMID:27461849

  14. Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

    ERIC Educational Resources Information Center

    Sui, Li; Wang, Jing; Li, Bao-Ming

    2008-01-01

    Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

  15. Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

    PubMed

    Elis, Winfried; Triantafellow, Ellen; Wolters, Natalie M; Sian, Katie R; Caponigro, Giordano; Borawski, Jason; Gaither, L Alex; Murphy, Leon O; Finan, Peter M; Mackeigan, Jeffrey P

    2008-04-01

    Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy. PMID:18403640

  16. Phosphoinositide 3-Kinase Gamma Contributes to Neuroinflammation in a Rat Model of Surgical Brain Injury

    PubMed Central

    Huang, Lei; Sherchan, Prativa; Wang, Yuechun; Reis, Cesar; Applegate, Richard L.; Tang, Jiping

    2015-01-01

    Neuroinflammation plays an important role in the pathophysiology of surgical brain injury (SBI). Phosphoinositide 3-kinase gamma (PI3Kγ), predominately expressed in immune and endothelial cells, activates multiple inflammatory responses. In the present study, we investigated the role of PI3Kγ and PI3Kγ-activated phosphodiesterase 3B (PDE3B) in neuroinflammation in a rat model of SBI. One hundred and fifty-two male Sprague Dawley rats (weight 280–350 g) were subjected to a partial right frontal lobe corticotomy model of SBI. A PI3Kγ pharmacological inhibitor (AS252424 or AS605240) was administered intraperitoneally. PI3Kγ siRNA, human recombinant active-PI3Kγ protein, or human recombinant active-PDE3B protein were administered intracerebroventricularly. Post-SBI assessments included neurobehavioral tests, brain water content, Western blot, and immunohistochemistry. Endogenous PI3Kγ levels were increased within peri-resection brain tissues after SBI, accompanied by increased brain water content and neurological functional deficits. There was a trend toward increased endogenous PDE3B phosphorylation after SBI. The selective PI3Kγ inhibitors AS252424 and AS605240 reduced brain water content surrounding corticotomy and improved neurological function after SBI. SBI increased and PI3Kγ inhibitor decreased levels of myeloperoxidase, cluster of differentiation 3, mast cell degranulation, E-selectin, and IL-1 in peri-resection brain tissues. Direct administration of human recombinant active-PI3Kγ protein and active-PDE3B protein countered the protective effect of AS252424. PI3Kγ siRNA reduced PI3Kγ levels, decreased brain water content within peri-resection brain tissues, and improved neurological function after SBI. Collectively, our findings suggest that PI3Kγ contributed to neuroinflammation after SBI. The use of selective PI3Kγ inhibitors may be a novel approach to ameliorating SBI via their anti-inflammation effects. SIGNIFICANCE STATEMENT Life-saving or

  17. Learning deficits and agenesis of synapses and myelinated axons in phosphoinositide-3 kinase-deficient mice.

    PubMed

    Tohda, Chihiro; Nakanishi, Ruiko; Kadowaki, Makoto

    Although previous studies have reported a role for phosphoinositide-3 kinase (PI3K) in axonal definition and growth in vitro, it is not clear whether PI3K regulates axonal formation and synaptogenesis in vivo. The goal of the present study was to clarify the role of PI3K in behavioral functions and some underlying neuroanatomical structures. Immunohistochemistry, an electron-microscopic analysis and behavioral tests were carried out. Knockout mice lacking the p85alpha regulatory subunit of PI3K (p85alpha-/- mice) significantly showed learning deficits, restlessness and motivation deficit. Expression of phosphorylated Akt, which indirectly shows the activity of PI3K, was high in myelinated axons, especially in axonal bundles in the striatum of wild-type mice, but was significantly low in the striatum, cerebral cortex and the hippocampal CA3 of p85alpha-/- mice. The axonal marker protein level decreased mainly in the striatum and cerebral cortex of p85alpha-/- mice. In these two regions, myelinated axons are rich in the wild-type mice. However, the density of myelinated axons and myelin thickness were significantly low in the striatum and cerebral cortex of p85alpha-/- mice. Synaptic protein level was clearly decreased in the striatum, cerebral cortex, and hippocampus of p85alpha-/- mice when compared with wild mice. The present results suggest that PI3K plays a role in the generation and/or maintenance of synapses and myelinated axons in the brain and that deficiencies in PI3K activity result in abnormalities in several neuronal functions, including learning, restlessness and motivation. PMID:17901711

  18. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA. PMID:19470781

  19. Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

    SciTech Connect

    Van Aller, Glenn S.; Carson, Jeff D.; Tang, Wei; Peng, Hao; Zhao, Lin; Copeland, Robert A.; Tummino, Peter J.; Luo, Lusong

    2011-03-11

    Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

  20. Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death.

    PubMed

    Lazo, John S; Sharlow, Elizabeth R; Epperly, Michael W; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M; Wipf, Peter; Greenberger, Joel S

    2013-12-01

    Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after

  1. Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

    PubMed Central

    Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.

    2013-01-01

    Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after

  2. Multiple forms of p55PIK, a regulatory subunit of phosphoinositide 3-kinase, are generated by alternative initiation of translation.

    PubMed Central

    Xia, X; Serrero, G

    1999-01-01

    A cDNA encoding p55PIK, one of the regulatory subunits of phosphoinositide (phosphatidylinositol) 3-kinase, was cloned from a cDNA library derived from the mouse mammary epithelial cell line C57MG. The cDNA coding for full-length p55PIK was transiently expressed in COS-7 cells. Western blot analysis of p55PIK expression using a specific antibody against p55PIK revealed that multiple protein products with different molecular masses were detected in COS-7 cell extracts. Experiments presented here demonstrate that multiple forms of p55PIK detected in COS-7 cells were produced by alternative initiation of translation. We also show that at least two in-frame start codons (AUG#2 and AUG#5) in p55PIK mRNA are used in COS-7 cells for the initiation of translation of p55PIK into proteins of 54 kDa and 50 kDa respectively. p55PIK mRNA was also alternatively translated into two proteins in PC cells, a mouse teratoma cell line, indicating that the alternative initiation of translation of p55PIK is not restricted to COS-7 cells. Results from immunoprecipitation and Western blot analysis showed that two forms (54 kDa and 50 kDa protein species) of p55PIK were detected in C57MG cells. Interestingly, when C57MG cells were treated with insulin, only p55PIK, but not p50PIK, bound to insulin receptor substrate-1 protein, providing evidence that different forms of p55PIKs may have specific distinct roles in signal transduction pathways. PMID:10417350

  3. Class I Phosphoinositide 3-Kinase Exerts a Differential Role on Cell Survival and Cell Trafficking in Retina.

    PubMed

    Azadi, Seifollah; Brush, Richard S; Anderson, Robert E; Rajala, Raju V S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides. They are responsible for coordinating a diverse range of cell functions including proliferation, cell survival, degranulation, vesicular trafficking, and cell migration. The PI 3-kinases are grouped into three distinct classes: I, II, and III. Class III PI3K has been shown to be involved in intracellular protein trafficking, whereas class I PI3K is known to regulate cell survival following activation of cell surface receptors. However, studies from our laboratory and others have shown that class I PI3K may also be involved in photoreceptor protein trafficking. Therefore, to learn more about the role of class I and class III P13K in trafficking and to understand the impact of the lipid content of trafficking cargo vesicles, we developed a methodology to isolate trafficking vesicles from retinal tissue. PI3K class I and III proteins were enriched in our extracted trafficking vesicle fraction. Moreover, levels of ether phosphatidylethanolamine (PE) and ether phosphatidylcholine (PC) were significantly higher in the trafficking vesicle fraction than in total retina. These two lipid classes have been suggested to be involved with fusion/targeting of trafficking vesicles. PMID:26427433

  4. Progress in the Preclinical Discovery and Clinical Development of Class I and Dual Class I/IV Phosphoinositide 3-Kinase (PI3K) Inhibitors

    PubMed Central

    Shuttleworth, S.J; Silva, F.A; Cecil, A.R.L; Tomassi, C.D; Hill, T.J; Raynaud, F.I; Clarke, P.A; Workman, P

    2011-01-01

    The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer. PMID:21649578

  5. Differential involvement of phosphoinositide 3-kinase in gonadotrophin-releasing hormone actions in gonadotrophs and somatotrophs of goldfish, Carassius auratus.

    PubMed

    Pemberton, Joshua G; Stafford, James L; Yu, Yi; Chang, John P

    2011-08-01

    In goldfish, two endogenous gonadotrophin-releasing hormones (GnRHs) [salmon (s)GnRH and chicken (c)GnRH-II] control maturational gonadotrophin-II [lutenising hormone (LH)] and growth hormone (GH) secretion via Ca(2+)-dependent intracellular signalling pathways. We investigated the involvement of phosphoinositide 3-kinase (PI3K) in GnRH-evoked LH and GH release and associated intracellular Ca(2+) increases ([Ca(2+)](i) ) in goldfish gonadotrophs and somatotrophs. Immunoreactive PI3K p85α, the predominant regulatory subunit for class IA PI3Ks, was detected in goldfish pituitary tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of pituitary cells and [Ca(2+)](i) increases in identified gonadotrophs were significantly reduced in the presence of PI3K inhibitors wortmannin (100 nm) and LY294002 (10 μm). Unexpectedly, wortmannin and LY294002 inhibited GnRH-evoked GH release but only attenuated the [Ca(2+)](i) response in identified somatotrophs to cGnRH-II, and not sGnRH. On the other hand, Ca(2+) ionophore-evoked LH and GH secretion remained unaltered in the presence of the PI3K inhibitors, suggesting that general decreases in the releasable hormone pool or sensitivity to [Ca(2+)](i) changes did not underlie the ability of wortmannin and LY294002 to reduce the actions of GnRH. These results provide the first evidence for the presence and involvement of PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. In gonadotrophs, the inhibitory action of PI3K on both sGnRH and cGnRH-II involves the attenuation of their evoked [Ca(2+)](i); in contrast, GnRH isoform-specific effects occur in somatotrophs. PMID:21649760

  6. Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers

    PubMed Central

    Kächele, Martin; Hennige, Anita M.; Machann, Jürgen; Hieronimus, Anja; Lamprinou, Apostolia; Machicao, Fausto; Schick, Fritz; Fritsche, Andreas; Stefan, Norbert; Nürnberg, Bernd; Häring, Hans-Ulrich; Staiger, Harald

    2015-01-01

    Objective Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis. Study Design Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis. Results After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs. Conclusions We could demonstrate that common genetic variation in the PIK3CG locus, possibly

  7. Dominant-Activating, Germline Mutations in Phosphoinositide 3-Kinase p110δ Cause T Cell Senescence and Human Immunodeficiency

    PubMed Central

    Lucas, Carrie L.; Kuehn, Hye Sun; Zhao, Fang; Niemela, Julie E.; Deenick, Elissa K.; Palendira, Umaimainthan; Avery, Danielle T.; Moens, Leen; Cannons, Jennifer L.; Biancalana, Matthew; Stoddard, Jennifer; Ouyang, Weiming; Frucht, David L.; Rao, V. Koneti; Atkinson, T. Prescott; Agharahimi, Anahita; Hussey, Ashleigh A.; Folio, Les R.; Olivier, Kenneth N.; Fleisher, Thomas A.; Pittaluga, Stefania; Holland, Steven M.; Cohen, Jeffrey I.; Oliviera, Joao B.; Tangye, Stuart G.; Schwartzberg, Pamela L.; Lenardo, Michael J.; Uzel, Gulbu

    2014-01-01

    The p110δ subunit of phosphoinositide 3-kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report three different germline, heterozygous, gain-of-function mutations in the PIK3CD gene encoding p110δ in fourteen patients from seven families. These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia. Strikingly, naïve and central memory T cells were severely deficient, while senescent effector T cells were over-represented. In vitro, patient T cells exhibited increased phosphorylation of Akt and hyperactivation of mTOR, enhanced glucose uptake and terminal effector differentiation. Importantly, treatment with rapamycin to inhibit mTOR activity in vivo partially restored naïve T cells, largely rescued the in vitro T cell defects, and improved clinical course. PMID:24165795

  8. Constitutive Macropinocytosis in Oncogene-transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3-Kinase and Phospholipase C

    PubMed Central

    Amyere, Mustapha; Payrastre, Bernard; Krause, Ulrike; Smissen, Patrick Van Der; Veithen, Alex; Courtoy, Pierre J.

    2000-01-01

    Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85α constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1,4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src–transformed cells for dominant-negative truncated p85α expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis. PMID:11029048

  9. Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

    PubMed

    Brando-Lima, Aline C; Saldanha-Gama, Roberta F; Henriques, Maria das Graças M O; Monteiro-Moreira, Ana C O; Moreira, Renato A; Barja-Fidalgo, Christina

    2005-10-15

    Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response. PMID:16183388

  10. The Structure of p85ni in Class IA Phosphoinositide 3-Kinase Exhibits Interdomain Disorder

    PubMed Central

    Sen, K. Ilker; Wu, Haiyan; Backer, Jonathan M.; Gerfen, Gary J.

    2010-01-01

    Regulation of the Class IA PI 3-kinase involves inhibition and stabilization of the catalytic subunit (p110) by the regulatory subunit (p85). Regulation is achieved by 2 major contacts: a stable interface involving the Adapter-Binding Domain (ABD) of p110 and the inter-SH2 (iSH2) domain of p85, and a regulatory interaction between the N-terminal SH2 (nSH2) domain of p85 and the helical domain of p110. In the present study, we have examined the relative orientation of the nSH2 and iSH2 of p85α using site-directed spin labeling and pulsed EPR. Surprisingly, both distance measurements and distance distributions suggest that the nSH2 domain is highly disordered relative to the iSH2 domain. Molecular modeling based on EPR distance restraints suggests that the nSH2 domain moves in a hinge-like manner, sampling a torus space around the proximal end of the iSH2 domain. These data have important implications for the mechanism by which p85/p110 dimers are regulated by phosphopeptides. PMID:20131869

  11. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

  12. Phosphoinositide 3-kinase dependent inhibition as a broad basis for opponent coding in Mammalian olfactory receptor neurons.

    PubMed

    Ukhanov, Kirill; Corey, Elizabeth A; Ache, Barry W

    2013-01-01

    Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs. PMID:23585911

  13. The PI 3-kinase and mTOR signaling pathways are important modulators of epithelial tubule formation.

    PubMed

    Walid, Shereaf; Eisen, Randi; Ratcliffe, Don R; Dai, Kezhi; Hussain, M Mahmood; Ojakian, George K

    2008-08-01

    Using MDCK cells as a model system, evidence is presented demonstrating that the signaling pathways mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI 3-kinase) play important roles in the regulation of epithelial tubule formation. Incubation of cells with collagen gel overlays induced early (4-8 h) reorganization of cells (epithelial remodeling) into three-dimensional multicellular tubular structures over 24 h. An MDCK cell line stably expressing the PH domain of Akt, a PI 3-kinase downstream effector, coupled to green fluorescent protein (GFP-Akt-PH) was used to determine the distribution of phosphatidyl inositol-3,4,5-P(3) (PIP(3)), a product of PI 3-kinase. GFP-Akt-PH was associated with lateral membranes in control cells. After incubation with collagen gel overlays, GFP-Akt-PH redistributed into the lamellipodia of migrating cells suggesting that PIP(3) plays a role in epithelial remodeling. Using the small molecule inhibitor LY-294002 that inhibits both mTOR and PI 3-kinase, we demonstrated that kinase activity was required for epithelial remodeling, disruption of cell junctions and subsequent modulation of tubule formation. Since the mTOR signaling pathway is downstream of PI 3-kinase, the effects of rapamycin, a specific mTOR inhibitor, on tubule formation were assessed. Rapamycin did not affect epithelial remodeling or GFP-Akt-PH redistribution but inhibited elongated tubule formation that occurred later (24 h) in morphogenesis. These results were further supported by using RNA interference to down-regulate mTOR and inhibit tubule formation. Our studies demonstrate that PI 3-kinase regulates early epithelial remodeling stages while mTOR modulates latter stages of tubule development. PMID:18366086

  14. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

    PubMed

    Tee, Andrew R; Anjum, Rana; Blenis, John

    2003-09-26

    The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. PMID:12867426

  15. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

    Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and

  16. Acquired PIK3CA amplification causes resistance to selective phosphoinositide 3-kinase inhibitors in breast cancer.

    PubMed

    Huw, L-Y; O'Brien, C; Pandita, A; Mohan, S; Spoerke, J M; Lu, S; Wang, Y; Hampton, G M; Wilson, T R; Lackner, M R

    2013-01-01

    Agents targeting the PI3K/mTOR signaling axis have shown promise in early-phase clinical trials and are currently being studied in later stages of clinical development in multiple indications. Experience with other targeted agents suggests that clinical responses may be short-lived because of acquired resistance to therapy. Here, we report preclinical modeling of acquired resistance in a HER2-positive, PIK3CA mutant breast cancer cell line, KPL-4. We identified a heretofore-unreported mechanism of resistance, specifically high-level amplification of the mutant allele of PIK3CA, which resulted in a marked upregulation of PI3K signaling, enabling resistant cells to regain proliferative capacity at clinically relevant concentrations of the PI3K inhibitor, GDC-0941. We show that knockdown of the amplified PIK3CA mutant allele in these cells by small interfering RNA restored pathway signaling and sensitivity to PI3K inhibition at levels comparable to parental cells. These novel preclinical findings suggest that, in addition to assessment of other previously reported mechanisms of resistance, evaluation of PI3K copy number variation should be integrated into the exploratory analysis of biopsies obtained at disease progression. PMID:24366379

  17. Single-Cell Analysis of Phosphoinositide 3-Kinase (PI3K) and Phosphatase and Tensin Homolog (PTEN) Activation

    PubMed Central

    Jiang, Dechen; Sims, Christopher Eldridge; Allbritton, Nancy Lynn

    2010-01-01

    Summary A single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (Bodipy Fl PIP2) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomoles. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that Bodipy Fl PIP2 was converted to phosphatidyl-inositol 3,4,5 trisphosphate (Bodipy Fl PIP3 ). Bodipy Fl PIP3 could be back converted to Bodipy Fl PIP2 by the phosphatase PTEN. When Bodipy Fl PIP2 was added to a cell lysate, 1.4 fmoles of the Bodipy Fl PIP3 were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of Bodipy Fl PIP3 to a cell lysate yielded 3 fmoles of Bodipy Fl PIP2 per ng of protein in 8 min. Both Bodipy Fl PIP2 and Bodipy Fl PIP3 were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the FcεR1 receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of Bodipy Fl PIP3 generated per cell increased 4-fold over that of unstimulated cells. This production of Bodipy Fl PIP3 was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level. PMID:21221426

  18. Hyperinsulinemia, glucose intolerance, and dyslipidemia induced by acute inhibition of phosphoinositide 3-kinase signaling in the liver.

    PubMed

    Miyake, Kazuaki; Ogawa, Wataru; Matsumoto, Michihiro; Nakamura, Takehiro; Sakaue, Hiroshi; Kasuga, Masato

    2002-11-01

    The physiological relevance of phosphoinositide 3-kinase (PI 3-K) signaling in the liver to fuel homeostasis was investigated. Systemic infusion of an adenovirus encoding a dominant negative mutant of PI 3-K ((Delta)p85) resulted in liver-specific expression of this protein and in inhibition of the insulin-induced activation of PI 3-K in the liver within 3 days, without affecting insulin signaling in skeletal muscle. Hepatic expression of (Delta)p85 led to hyperinsulinemia and to a marked increase in blood glucose concentration in response to oral glucose intake. The increases in both glycogen and glucose 6-phosphate content, as well as in Akt and glycogen synthase activities in the liver, that were induced by glucose intake were markedly impaired in mice expressing (Delta)p85. Despite an upregulation of mRNAs for gluconeogenic enzymes apparent in the liver of these animals, the fasting blood glucose concentration was increased only slightly, and the serum concentrations of gluconeogenic precursors were reduced. However, administration of pyruvate, a substrate for gluconeogenesis, resulted in an exaggerated increase in blood glucose concentration. In the fasted state, the mass of adipose tissue of the mice was about 1.5 times that in control mice. The mice also exhibited marked decreases in the serum concentrations of FFAs and triglyceride and suppression of insulin-induced PI 3-K activation in adipose tissue, probably due to the associated hyperinsulinemia. PI 3-K activity in the liver is thus essential for normal carbohydrate and lipid metabolism in living animals. PMID:12438446

  19. Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration.

    PubMed

    Horiguchi, Michiko; Oiso, Yuki; Sakai, Hitomi; Motomura, Tomoki; Yamashita, Chikamasa

    2015-09-10

    Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causing widespread and irreversible alveoli collapse. The discovery of a low-molecular-weight compound that induces regeneration of pulmonary alveoli is of utmost urgency to cure intractable pulmonary diseases such as COPD. However, a practically useful compound for regenerating pulmonary alveoli is yet to be reported. Previously, we have elucidated that Akt phosphorylation is involved in a differentiation-inducing molecular mechanism of human alveolar epithelial stem cells, which play a role in regenerating pulmonary alveoli. In the present study, we directed our attention to phosphoinositide 3-kinase (PI3K)-Akt signaling and examined whether PI3K inhibitors display the pulmonary alveolus regeneration. Three PI3K inhibitors with different PI3K subtype specificities (Wortmannin, AS605240, PIK-75 hydrochloride) were tested for the differentiation-inducing effect on human alveolar epithelial stem cells, and Wortmannin demonstrated the most potent differentiation-inducing activity. We evaluated Akt phosphorylation in pulmonary tissues of an elastase-induced murine COPD model and found that Akt phosphorylation in the pulmonary tissue was enhanced in the murine COPD model compared with normal mice. Then, the alveolus-repairing effect of pulmonary administration of Wortmannin to murine COPD model was evaluated using X-ray CT analysis and hematoxylin-eosin staining. As a result, alveolar damages were repaired in the Wortmannin-administered group to a similar level of normal mice. Furthermore, pulmonary administration of Wortmannin induced a significant recovery of the respiratory function, compared to the control group. These results indicate that Wortmannin is capable of inducing differentiation of human alveolar epithelial stem cells and represents a promising drug candidate for curative treatment of pulmonary alveolar destruction in COPD. PMID:26160307

  20. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines

    PubMed Central

    Engelman, Jeffrey A.; Jänne, Pasi A.; Mermel, Craig; Pearlberg, Joseph; Mukohara, Toru; Fleet, Christina; Cichowski, Karen; Johnson, Bruce E.; Cantley, Lewis C.

    2005-01-01

    Therapies that target the EGF receptor (EGFR), such as gefitinib (IRESSA), are effective in a subset of patients with advanced non-small cell lung cancer (NSCLC). The differences in intracellular signaling networks between gefitinib-sensitive and -resistant NSCLCs remain poorly understood. In this study, we observe that gefitinib reduces phospho-Akt levels only in NSCLC cell lines in which it inhibits growth. To elucidate the mechanism underlying this observation, we compared immunoprecipitates of phosphoinositide 3-kinase (PI3K) between gefitinib-sensitive and -resistant NSCLC cell lines. We observe that PI3K associates with ErbB-3 exclusively in gefitinib-sensitive NSCLC cell lines. Gefitinib dissociates this complex, thereby linking EGFR inhibition to decreased Akt activity. In contrast, gefitinib-resistant cells do not use ErbB-3 to activate the PI3K/Akt pathway. In fact, abundant ErbB-3 expression is detected only in gefitinib-sensitive NSCLC cell lines. Two gefitinib-sensitive NSCLC cell lines with endogenous distinct activating EGFR mutations (L858R and Del747-749), frequently observed in NSCLC patients who respond to gefitinib, also use ErbB-3 to couple to PI3K. Down-regulation of ErbB-3 by means of short hairpin RNA leads to decreased phospho-Akt levels in the gefitinib-sensitive NSCLC cell lines, Calu-3 (WT EGFR) and H3255 (L858R EGFR), but has no effect on Akt activation in the gefitinib-resistant cell lines, A549 and H522. We conclude that ErbB-3 is used to couple EGFR to the PI3K/Akt pathway in gefitinib-sensitive NSCLC cell lines harboring WT and mutant EGFRs. PMID:15731348

  1. Phosphatidylinositol 3-kinase pathway activation in breast cancer brain metastases

    PubMed Central

    2011-01-01

    Introduction Activation status of the phosphatidylinositol 3-kinase (PI3K) pathway in breast cancer brain metastases (BCBMs) is largely unknown. We examined expression of phospho(p)-AKT, p-S6, and phosphatase and tensin homologue (PTEN) in BCBMs and their implications for overall survival (OS) and survival after BCBMs. Secondary analyses included PI3K pathway activation status and associations with time to distant recurrence (TTDR) and time to BCBMs. Similar analyses were also conducted among the subset of patients with triple-negative BCBMs. Methods p-AKT, p-S6, and PTEN expression was assessed with immunohistochemistry in 52 BCBMs and 12 matched primary BCs. Subtypes were defined as hormone receptor (HR)+/HER2-, HER2+, and triple-negative (TNBC). Survival analyses were performed by using a Cox model, and survival curves were estimated with the Kaplan-Meier method. Results Expression of p-AKT and p-S6 and lack of PTEN (PTEN-) was observed in 75%, 69%, and 25% of BCBMs. Concordance between primary BCs and matched BCBMs was 67% for p-AKT, 58% for p-S6, and 83% for PTEN. PTEN- was more common in TNBC compared with HR+/HER2- and HER2+. Expression of p-AKT, p-S6, and PTEN- was not associated with OS or survival after BCBMs (all, P > 0.06). Interestingly, among all patients, PTEN- correlated with shorter time to distant and brain recurrence. Among patients with TNBC, PTEN- in BCBMs was associated with poorer overall survival. Conclusions The PI3K pathway is active in most BCBMs regardless of subtype. Inhibition of this pathway represents a promising therapeutic strategy for patients with BCBMs, a group of patients with poor prognosis and limited systemic therapeutic options. Although expression of the PI3K pathway did not correlate with OS and survival after BCBM, PTEN- association with time to recurrence and OS (among patients with TNBC) is worthy of further study. PMID:22132754

  2. 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

  3. Illuminating the phosphatidylinositol 3-kinase/Akt pathway

    NASA Astrophysics Data System (ADS)

    Ni, Qiang; Fosbrink, Matthew; Zhang, Jin

    2008-02-01

    Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined nuclear phosphatidylinositol-3,4,5-triphosphate (PIP 3) in living cells using nucleus-targeted InPAkt. Second, we analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and nucleus-targeted BKAR to simultaneously monitor PIP 3 dynamics and Akt activity in the same cell. Of note, results from these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the nucleus in the presence of high levels of PIP 3 at the plasma membrane. This finding has led to a further study of the action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

  4. Chromosomal Instability and Phosphoinositide Pathway Gene Signatures in Glioblastoma Multiforme.

    PubMed

    Waugh, Mark G

    2016-01-01

    Structural rearrangements of chromosome 10 are frequently observed in glioblastoma multiforme and over 80 % of tumour samples archived in the catalogue of somatic mutations in cancer database had gene copy number loss for PI4K2A which encodes phosphatidylinositol 4-kinase type IIalpha. PI4K2A loss of heterozygosity mirrored that of PTEN, another enzyme that regulates phosphoinositide levels and also PIK3AP1, MINPP1, INPP5A and INPP5F. These results indicated a reduction in copy number for a set of phosphoinositide signalling genes that co-localise to chromosome 10q. This analysis was extended to a panel of phosphoinositide pathway genes on other chromosomes and revealed a number of previously unreported associations with glioblastoma multiforme. Of particular note were highly penetrant copy number losses for a group of X-linked phosphoinositide phosphatase genes OCRL, MTM1 and MTMR8; copy number amplifications for the chromosome 19 genes PIP5K1C, AKT2 and PIK3R2, and also for the phospholipase C genes PLCB1, PLCB4 and PLCG1 on chromosome 20. These mutations are likely to affect signalling and trafficking functions dependent on the PI(4,5)P2, PI(3,4,5)P3 and PI(3,5)P2 lipids as well as the inositol phosphates IP3, IP5 and IP6. Analysis of flanking genes with functionally unrelated products indicated that chromosomal instability as opposed to a phosphoinositide-specific process underlay this pattern of copy number variation. This in silico study suggests that in glioblastoma multiforme, karyotypic changes have the potential to cause multiple abnormalities in sets of genes involved in phosphoinositide metabolism and this may be important for understanding drug resistance and phosphoinositide pathway redundancy in the advanced disease state. PMID:25502460

  5. The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons

    PubMed Central

    Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

    2008-01-01

    Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-γ-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

  6. Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars.

    PubMed

    Liu, Hong-Wei; Cheng, Biao; Yu, Wen-Lin; Sun, Rui-Xia; Zeng, Dong; Wang, Jie; Liao, Yuan-Xing; Fu, Xiao-Bing

    2006-06-27

    Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and production of extracellular matrix, which are very important process in skin wound healing and scarring; however, the signaling pathways involved in this process, especially in humans, are less explored. In the present study, we used skin fibroblasts of human hypertrophic scar, which expressed both AT1 and AT2 receptors, and observed that Ang II increased Akt phosphorylation and phosphoinositide 3 kinase (PI 3-K) activity. In addition, the Ang II-induced Akt phosphorylation was blocked by wortmannin, a PI 3-K inhibitor. This Ang II-activated PI 3-K/Akt cascade was markedly inhibited by valsartan, an AT(1) receptor-specific blocker, whereas it was enhanced by PD123319, an AT(2) receptor antagonist. On the other hand, the Ang II- or EGF-induced activation of PI 3-K/Akt was strongly attenuated by AG1478, an inhibitor of epidermal growth factor (EGF) receptor kinase. Moreover, Ang II stimulated tyrosine phosphorylation of EGF receptor and p85alpha subunit of PI 3-K accompanied by an increase in their association, which was inhibited by valsartan, and enhanced by PD123319. The Ang II-induced transactivation of EGF receptor resulted in activation of extracellular signal-regulated kinase (ERK) that was also inhibited by valsartan, and enhanced by PD123319. Taken together, our results showed that AT(1) receptor-mediated activation of PI 3-K/Akt cascades occurs at least partially via the transactivation of EGF receptor, which is under a negative control by AT(2) receptor in hypertrophic scar fibroblasts. These findings contribute to understanding the molecular mechanism of human hypertrophic scar formation. PMID:16522324

  7. Apelin/APJ signaling promotes hypoxia-induced proliferation of endothelial progenitor cells via phosphoinositide-3 kinase/Akt signaling.

    PubMed

    Zhang, Jingchang; Liu, Qiming; Hu, Xinqun; Fang, Zhenfei; Huang, Feng; Tang, Liang; Zhou, Shenghua

    2015-09-01

    Endothelial progenitor cells (EPCs) can adhere to the endothelium at sites of hypoxia/ischemia and participate in the formation of novel vessels through differentiating into endothelial cells (ECs). Apelin is an endogenous ligand for the G protein‑coupled receptor APJ, and apelin/APJ signaling has a role in cardiovascular function. The present study aimed to investigate the role of apelin/APJ signaling in the regulation of EPC proliferation under hypoxia. The results showed that hypoxia was able to induce EPC proliferation, accompanied with an upregulation of hypoxia‑inducible factor (HIF)‑1α as well as apelin/APJ signaling. Further investigation indicated that siRNA‑mediated knockdown of apelin or APJ expression attenuated the hypoxia‑induced proliferation of EPCs, suggesting that apelin/APJ signaling has an important role in hypoxia‑induced EPC proliferation. Moreover, the phosphoinositide‑3 kinase (PI3K)/Akt signaling pathway was found to be involved in the apelin/APJ‑mediated EPC proliferation under hypoxia. Based on these findings, the present study suggested that hypoxia‑induced upregulation of HIF‑1α promotes the expression of apelin and APJ, which further activate the downstream PI3K/Akt signaling pathway, a key promoter of EPC proliferation. In conclusion, the present study highlighted the role of apelin/APJ in the regulation of EPC proliferation, and apelin/APJ may therefore serve as a potential target for the prevention of hypoxic ischemic injury. PMID:26018184

  8. PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    PubMed Central

    Tsuchiya, A; Kanno, T; Nishizaki, T

    2014-01-01

    Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1 nM–1 μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation. PMID:24169049

  9. Clionosterol and ethyl cholestan-22-enol isolated from the rhizome of Polygala tenuifolia inhibit phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Le, Thi Kim Van; Jeong, Jin Ju; Kim, Dong-Hyun

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors were isolated from the rhizome of Polygala tenuifolia WILLD (PT, Polygalaceae), which has been used in traditional Chinese medicine for inflammation, dementia, amnesia, neurasthenia and cancer, by activity-guided fractionation. For the assay of PI3K/Akt pathway, cytoprotective Tat-transduced CHME5 cells, which are the cytoprotective phenotype against lypopolysaccharide (LPS)/cycloheximide (CHX), were used. We isolated 4 anti-cytoprotective compounds, clionasterol (1), ethyl cholestan-22-enol (2), 3-O-β-D-glucosyl ethyl cholestan-22-enol (3), and 3-O-β-D-glucopyranosyl clionasterol (4) from EtOAc fraction of PT against Tat-transduced CHME5 cells. Of them, (1) and (2) most potently abolished cytoprotective effect of Tat-transduced CHME5 cells. These constituents (1) and (2) inhibited the activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and its downstream molecules, Akt/glycogen synthase kinase (GSK)3β, in PI3K/Akt cell survival signaling pathway, but did not suppress the activation of PI3K. Based on these finding, (1) and (2) may abolish the cytoprotective phenotype of Tat-transduced CHME5 cells by inhibiting PDK1 phosphorylation in PI3K/Akt pathway. PMID:22863942

  10. 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

  11. Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases.

    PubMed

    Vaidhyanathan, Shruthi; Wilken-Resman, Brynna; Ma, Daniel J; Parrish, Karen E; Mittapalli, Rajendar K; Carlson, Brett L; Sarkaria, Jann N; Elmquist, William F

    2016-02-01

    Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood-brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood-brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show that

  12. Molecular Dynamics Simulations to Investigate the Binding Mode of the Natural Product Liphagal with Phosphoinositide 3-Kinase α.

    PubMed

    Gao, Yanjuan; Ma, Ying; Yang, Guangde; Li, Yiping

    2016-01-01

    Phosphatidylinositol 3-kinase α (PI3Kα) is an attractive target for anticancer drug design. Liphagal, isolated from the marine sponge Aka coralliphaga, possesses the special "liphagane" meroterpenoid carbon skeleton and has been demonstrated as a PI3Kα inhibitor. Molecular docking and molecular dynamics simulations were performed to explore the dynamic behaviors of PI3Kα binding with liphagal, and free energy calculations and energy decomposition analysis were carried out by use of molecular mechanics/Poisson-Boltzmann (generalized Born) surface area (MM/PB(GB)SA) methods. The results reveal that the heteroatom rich aromatic D-ring of liphagal extends towards the polar region of the binding site, and the D-ring 15-hydroxyl and 16-hydroxyl form three hydrogen bonds with Asp810 and Tyr836. The cyclohexyl A-ring projects up into the upper pocket of the lipophilic region, and the hydrophobic/van der Waals interactions with the residues Met772, Trp780, Ile800, Ile848, Val850, Met922, Phe930, Ile932 could be the key interactions for the affinity of liphagal to PI3Kα. Thus, a new strategy for the rational design of more potent analogs of liphagal against PI3Kα is provided. Our proposed PI3Kα/liphagal binding mode would be beneficial for the discovery of new active analogs of liphagal against PI3Kα. PMID:27367663

  13. Genetic or Pharmaceutical Blockade of Phosphoinositide 3-Kinase P110δ Prevents Chronic Rejection of Heart Allografts

    PubMed Central

    Rose, Marlene L.; McCormack, Ann M.; Sarathchandra, Padmini; Okkenhaug, Klaus; Marelli-Berg, Federica M.

    2012-01-01

    Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans. PMID:22479345

  14. Definition of the binding mode of phosphoinositide 3-kinase α-selective inhibitor A-66S through molecular dynamics simulation.

    PubMed

    Bian, Xiaoli; Dong, Wangqing; Zhao, Yang; Sun, Rui; Kong, Wanjun; Li, Yiping

    2014-04-01

    Activation of the phosphatidylinositol 3-kinase α (PI3Kα) is commonly observed in human cancer and is critical for tumor progression, which has made PI3Kα an attractive target for anticancer drug discovery. To systematically investigate the binding mode of A-66S, a new selective PI3Kα inhibitor for PI3Kα, molecular docking, molecular dynamics simulation and ensuing energetic analysis were performed. The binding free energy between PI3Kα and A-66S is -11.27 kcal•mol⁻¹ using MMPBSA method, while -14.67 kcal•mol⁻¹ using MMGBSA method, which is beneficial for the binding, and the van der Waals/hydrophobic and electrostatic interactions are critical for the binding. The conserved hydrophobic adenine region of PI3Kα made up of Met772, Pro778, Ile800, Tyr836, Ile848, Val850, Val851, Met922, Phe930 and Ile932 accommodates the flat 2-tert-butyl-4'-methyl-4,5'-bithiazol moiety of A-66S, and the NH of Val851 forms a hydrogen with the nitrogen atom embedded in the aminothiazole ring of A-66S. The (S)-pyrrolidine carboxamide urea moiety especially extends toward the region of the binding site wall (Ser854-Gln859) defined by the C-terminal lobe, and has three hydrogen-bond arms with the backbone of Ser854 and the side chain of Gln859. Notably the interaction between the non-conserved residue Gln859 and A-66S is responsible for the selectivity profile of A-66S. The binding mode of A-66S for PI3Kα presented in this study should aid in the design of a new highly selective PI3Kα inhibitor. PMID:24633771

  15. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins

    PubMed Central

    Schlam, Daniel; Bagshaw, Richard D.; Freeman, Spencer A.; Collins, Richard F.; Pawson, Tony; Fairn, Gregory D.; Grinstein, Sergio

    2015-01-01

    Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large—but not small—phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles. PMID:26465210

  16. miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma

    SciTech Connect

    Chen, Suling; Li, Fang; Chai, Haiyun; Tao, Xin; Wang, Haili; Ji, Aifang

    2015-08-21

    MicroRNAs (miRNAs) play a key role in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). In the present study, we demonstrated that miR-502 significantly inhibits HCC cell proliferation in vitro and tumor growth in vivo. G1/S cell cycle arrest and apoptosis of HCC cells were induced by miR-502. Phosphoinositide 3-kinase catalytic subunit gamma (PIK3CG) was identified as a direct downstream target of miR-502 in HCC cells. Notably, overexpression of PIK3CG reversed the inhibitory effects of miR-502 in HCC cells. Our findings suggest that miR-502 functions as a tumor suppressor in HCC via inhibition of PI3KCG, supporting its utility as a promising therapeutic gene target for this tumor type. - Highlights: • miR-502 suppresses HCC cell proliferation in vitro and tumorigenicity in vivo. • miR-502 regulates cell cycle and apoptosis in HCC cells. • PIK3CG is a direct target of miR-502. • miR-502 and PIK3CG expression patterns are inversely correlated in HCC tissues.

  17. Salinomycin causes migration and invasion of human fibrosarcoma cells by inducing MMP-2 expression via PI3-kinase, ERK-1/2 and p38 kinase pathways.

    PubMed

    Yu, Seon-Mi; Kim, Song Ja

    2016-06-01

    Salinomycin (SAL) is a polyether ionophore antibiotic that has recently been shown to regulate a variety of cellular responses in various human cancer cells. However, the effects of SAL on metastatic capacity of HT1080 human fibrosarcoma cells have not been elucidated. We investigated the effect of SAL on migration and invasion, with emphasis on the expression and activation of matrix metalloproteinase (MMP)-2 in HT1080 human fibrosarcoma cells. Treatment of SAL promoted the expression and activation of MMP-2 in a dose- and time-dependent manner, as detected by western blot analysis, gelatin zymography, and real-time polymerase chain reaction. SAL also increased metastatic capacities, as determined by an increase in the migration and invasion of cells using the wound healing assay and the invasion assay, respectively. To confirm the detailed molecular mechanisms of these effects, we measured the activation of phosphoinositide 3 kinase (PI3-kinase) and mitogen-activated protein kinase (MAPK)s (ERK-1/2 and p38 kinase), as detected by the phosphorylated proteins through western blot analysis. SAL treatment increased the phosphorylation of Akt and MAPKs. Inhibition of PI3-kinase, ERK-1/2, and p38 kinase with LY294002, PD98059, and SB203580, respectively, in the presence of SAL suppressed the metastatic capacity by reducing MMP-2 expression, as determined by gelatin zymography. Our results indicate that the PI3-kinase and MAPK signaling pathways are involved in migration and invasion of HT1080 through induction of MMP-2 expression and activation. In conclusion, SAL significantly increases the metastatic capacity of HT1080 cells by inducing MMP-2 expression via PI3-kinase and MAPK pathways. Our results suggest that SAL may be a potential agent for the study of cancer metastatic capacities. PMID:27035160

  18. Imidazo[1,2-a]pyrazine inhibitors of phosphoinositide 3-kinase alpha (PI3Kα): 3D-QSAR analysis utilizing the Hybrid Monte Carlo algorithm to refine receptor-ligand complexes for molecular alignment.

    PubMed

    Chadha, N; Jasuja, H; Kaur, M; Singh Bahia, M; Silakari, O

    2014-01-01

    Phosphoinositide 3-kinase alpha (PI3Kα) is a lipid kinase involved in several cellular functions such as cell growth, proliferation, differentiation and survival, and its anomalous regulation leads to cancerous conditions. PI3Kα inhibition completely blocks the cancer signalling pathway, hence it can be explored as an important therapeutic target for cancer treatment. In the present study, docking analysis of 49 selective imidazo[1,2-a]pyrazine inhibitors of PI3Kα was carried out using the QM-Polarized ligand docking (QPLD) program of the Schrödinger software, followed by the refinement of receptor-ligand conformations using the Hybrid Monte Carlo algorithm in the Liaison program, and alignment of refined conformations of inhibitors was utilized for the development of an atom-based 3D-QSAR model in the PHASE program. Among the five generated models, the best model was selected corresponding to PLS factor 2, displaying the highest value of Q(2)test (0.650). The selected model also displayed high values of r(2)train (0.917), F-value (166.5) and Pearson-r (0.877) and a low value of SD (0.265). The contour plots generated for the selected 3D-QSAR model were correlated with the results of docking simulations. Finally, this combined information generated from 3D-QSAR and docking analysis was used to design new congeners. PMID:24601789

  19. Phosphoinositide 3-Kinase γ Protects Against Catecholamine-Induced Ventricular Arrhythmia Through Protein Kinase A–Mediated Regulation of Distinct Phosphodiesterases

    PubMed Central

    Ghigo, Alessandra; Perino, Alessia; Mehel, Hind; Zahradníková, Alexandra; Morello, Fulvio; Leroy, Jérôme; Nikolaev, Viacheslav O.; Damilano, Federico; Cimino, James; De Luca, Elisa; Richter, Wito; Westenbroek, Ruth; Catterall, William A.; Zhang, Jin; Yan, Chen; Conti, Marco; Gomez, Ana Maria; Vandecasteele, Grégoire; Hirsch, Emilio; Fischmeister, Rodolphe

    2014-01-01

    Background Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by β-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown. Methods and Results Mice lacking PI3Kγ (PI3Kγ−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective β2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after β2-adrenergic receptor activation in PI3Kγ−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from β-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3Kγ−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia. PMID:23008439

  20. Hepatoprotective Effect of Quercetin on Endoplasmic Reticulum Stress and Inflammation after Intense Exercise in Mice through Phosphoinositide 3-Kinase and Nuclear Factor-Kappa B

    PubMed Central

    Tang, Yuhan; Li, Juan; Gao, Chao; Xu, Yanyan; Li, Yanyan; Yu, Xiao; Wang, Jing; Liu, Liegang

    2016-01-01

    The mechanisms underlying intense exercise-induced liver damage and its potential treatments remain unclear. We explored the hepatoprotection and mechanisms of quercetin, a naturally occurring flavonoid, in strenuous exercise-derived endoplasmic reticulum stress (ERS) and inflammation. Intense exercise (28 m/min at a 5° slope for 90 min) resulted in the leakage of aminotransferases in the BALB/C mice. The hepatic ultrastructural malformations and oxidative stress levels were attenuated by quercetin (100 mg/kg·bw). Intense exercise and thapsigargin- (Tg-) induced ERS (glucose-regulated protein 78, GRP78) and inflammatory cytokines levels (IL-6 and TNF-α) were decreased with quercetin. Furthermore, quercetin resulted in phosphoinositide 3-kinase (PI3K) induction, Ca2+ restoration, and blockade of the activities of Jun N-terminal kinase (JNK), activating transcription factor 6 (ATF6) and especially NF-κB (p65 and p50 nuclear translocation). A PI3K inhibitor abrogated the protection of quercetin on ERS and inflammation of mouse hepatocytes. SP600125 (JNK inhibitor), AEBSF (ATF6 inhibitor), and especially PDTC (NF-κB inhibitor) enhanced the quercetin-induced protection against Tg stimulation. Collectively, intense exercise-induced ERS and inflammation were attenuated by quercetin. PI3K/Akt activation and JNK, ATF6, and especially NF-κB suppression were involved in the protection. Our results highlight a novel preventive strategy for treating ERS and inflammation-mediated liver damage induced by intense exercise using natural phytochemicals. PMID:27504150

  1. Kinase-dependent and -independent functions of the p110β phosphoinositide-3-kinase in cell growth, metabolic regulation and oncogenic transformation

    PubMed Central

    Jia, Shidong; Liu, Zhenning; Zhang, Sen; Liu, Pixu; Zhang, Lei; Lee, Sang Hyun; Zhang, Jing; Signoretti, Sabina; Loda, Massimo; Roberts, Thomas M.; Zhao, Jean J.

    2009-01-01

    Upon activation by receptors, the ubiquitously expressed Class IA isoforms (p110α and p110β) of phosphoinositide-3-kinase (PI3K) generate lipid second messengers, which initiate multiple signal transduction cascades1–5. Recent studies have demonstrated specific roles for p110α in growth factor and insulin signaling6–8. To probe for distinct functions of p110β, we constructed conditional knockout mice. Ablation of p110β in the livers of the resulting mice led to impaired insulin sensitivity and glucose homeostasis, while having little effect on Akt-phosphorylation, suggesting involvement of a kinase-independent role of p110β in insulin metabolic action. Using established mouse embryonic fibroblasts (MEFs), we found that removal of p110β also had little effect on Akt-phosphorylation in response to insulin and EGF stimulation, but resulted in retarded cell proliferation. Reconstitution of p110β-null cells with a wild-type or kinase-dead allele of p110β demonstrated that p110β possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110β was required for LPA triggered GPCR signalling and played a role in oncogenic transformation. Most strikingly, in an animal model of prostate tumor formation induced by PTEN loss, ablation of p110β, but not p110α, impeded tumorigenesis with concomitant diminution of Akt-phosphorylation. Taken together our findings demonstrate both kinase-dependent and -independent functions for p110β, and strongly point to the kinase-dependent functions of p110β as a promising target in cancer therapy. PMID:18594509

  2. Hepatoprotective Effect of Quercetin on Endoplasmic Reticulum Stress and Inflammation after Intense Exercise in Mice through Phosphoinositide 3-Kinase and Nuclear Factor-Kappa B.

    PubMed

    Tang, Yuhan; Li, Juan; Gao, Chao; Xu, Yanyan; Li, Yanyan; Yu, Xiao; Wang, Jing; Liu, Liegang; Yao, Ping

    2016-01-01

    The mechanisms underlying intense exercise-induced liver damage and its potential treatments remain unclear. We explored the hepatoprotection and mechanisms of quercetin, a naturally occurring flavonoid, in strenuous exercise-derived endoplasmic reticulum stress (ERS) and inflammation. Intense exercise (28 m/min at a 5° slope for 90 min) resulted in the leakage of aminotransferases in the BALB/C mice. The hepatic ultrastructural malformations and oxidative stress levels were attenuated by quercetin (100 mg/kg·bw). Intense exercise and thapsigargin- (Tg-) induced ERS (glucose-regulated protein 78, GRP78) and inflammatory cytokines levels (IL-6 and TNF-α) were decreased with quercetin. Furthermore, quercetin resulted in phosphoinositide 3-kinase (PI3K) induction, Ca(2+) restoration, and blockade of the activities of Jun N-terminal kinase (JNK), activating transcription factor 6 (ATF6) and especially NF-κB (p65 and p50 nuclear translocation). A PI3K inhibitor abrogated the protection of quercetin on ERS and inflammation of mouse hepatocytes. SP600125 (JNK inhibitor), AEBSF (ATF6 inhibitor), and especially PDTC (NF-κB inhibitor) enhanced the quercetin-induced protection against Tg stimulation. Collectively, intense exercise-induced ERS and inflammation were attenuated by quercetin. PI3K/Akt activation and JNK, ATF6, and especially NF-κB suppression were involved in the protection. Our results highlight a novel preventive strategy for treating ERS and inflammation-mediated liver damage induced by intense exercise using natural phytochemicals. PMID:27504150

  3. Dual inhibition of histone deacetylases and phosphoinositide 3-kinases: effects on Burkitt lymphoma cell growth and migration.

    PubMed

    Ferreira, Ana Carolina dos Santos; de-Freitas-Junior, Julio Cesar Madureira; Morgado-Díaz, Jose Andres; Ridley, Anne J; Klumb, Claudete Esteves

    2016-04-01

    Burkitt lymphoma is a highly aggressive non-Hodgkin lymphoma that is characterized by MYC deregulation. Recently, the PI3K pathway has emerged as a cooperative prosurvival mechanism in Burkitt lymphoma. Despite the highly successful results of treatment that use high-dose chemotherapy regimens in pediatric Burkitt lymphoma patients, the survival rate of pediatric patients with progressive or recurrent disease is low. PI3Ks are also known to regulate cell migration, and abnormal cell migration may contribute to cancer progression and dissemination in Burkitt lymphoma. Little is known about Burkitt lymphoma cell migration, but the cooperation between MYC and PI3K in Burkitt lymphoma pathogenesis suggests that a drug combination could be used to target the different steps involved in Burkitt lymphoma cell dissemination and disease progression. The aim of this study was to investigate the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid combined with the PI3K inhibitor LY294002 on Burkitt lymphoma cell growth and migration. The combination enhanced the cell growth inhibition and cell-cycle arrest induced by the PI3K inhibitor or histone deacetylase inhibitor individually. Moreover, histone deacetylase inhibitor/PI3K inhibitor cotreatment suppressed Burkitt lymphoma cell migration and decreased cell polarization, Akt and ERK1/2 phosphorylation, and leads to RhoB induction. In summary, the histone deacetylase inhibitor/PI3Ki combination inhibits cell proliferation and migration via alterations in PI3K signaling and histone deacetylase activity, which is involved in the acetylation of α-tubulin and the regulation of RhoB expression. PMID:26561567

  4. Apoptosis and inactivation of the PI3-kinase pathway by tetrocarcin A in breast cancers

    SciTech Connect

    Nakajima, Hiroo; Sakaguchi, Koichi; Fujiwara, Ikuya; Mizuta, Mitsuhiko; Tsuruga, Mie; Magae, Junji . E-mail: jmagae@sannet.ne.jp; Mizuta, Naruhiko

    2007-04-27

    A survival kinase, Akt, is a downstream factor in the phosphatidylinositide-3'-kinase-dependent pathway, which mediates many biological responses including glucose uptake, protein synthesis and the regulation of proliferation and apoptosis, which is assumed to contribute to acquisition of malignant properties of human cancers. Here we find that an anti-tumor antibiotic, tetrocarcin A, directly induces apoptosis of human breast cancer cells. The apoptosis is accompanied by the activation of a proteolytic cascade of caspases including caspase-3 and -9, and concomitantly decreases phosphorylation of Akt, PDK1, and PTEN, a tumor suppressor that regulates the activity of Akt through the dephosphorylation of polyphosphoinositides. Tetrocarcin A affected neither expression of Akt, PDK1, or PTEN, nor did it affect the expression of Bcl family members including Bcl-2, Bcl-X{sub L}, and Bax. These results suggest that tetrocarcin A could be a potent chemotherapeutic agent for human breast cancer targeting the phosphatidylinositide-3'-kinase/Akt signaling pathway.

  5. Role of Phosphatidylinositol-3-Kinase Pathway in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Du, Li; Shen, Jingping; Weems, Andrew; Lu, Shi-Long

    2012-01-01

    Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most frequently observed molecular alterations in many human malignancies, including head and neck squamous cell carcinoma (HNSCC). A growing body of evidence demonstrates the prime importance of the PI3K pathway at each stage of tumorigenesis, that is, tumor initiation, progression, recurrence, and metastasis. Expectedly, targeting the PI3K pathway yields some promising results in both preclinical studies and clinical trials for certain cancer patients. However, there are still many questions that need to be answered, given the complexity of this pathway and the existence of its multiple feedback loops and interactions with other signaling pathways. In this paper, we will summarize recent advances in the understanding of the PI3K pathway role in human malignancies, with an emphasis on HNSCC, and discuss the clinical applications and future direction of this field. PMID:22666248

  6. Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

    PubMed Central

    Sohara, Yasuyoshi; Ishiguro, Naoki; Machida, Kazuya; Kurata, Hisashi; Thant, Aye Aye; Senga, Takeshi; Matsuda, Satoru; Kimata, Koji; Iwata, Hisashi; Hamaguchi, Michinari

    2001-01-01

    We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells. PMID:11408591

  7. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

    PubMed

    Aragoneses-Fenoll, L; Montes-Casado, M; Ojeda, G; Acosta, Y Y; Herranz, J; Martínez, S; Blanco-Aparicio, C; Criado, G; Pastor, J; Dianzani, U; Portolés, P; Rojo, J M

    2016-04-15

    Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis. PMID:26883061

  8. 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

  9. PI-3 kinase pathway can mediate the effect of TGF-beta1 in inducing the expression of SHARP-2 in LLC-PK1 cells.

    PubMed

    Shou, Zhang-fei; Zhou, Qin; Cai, Jie-ru; Chen, Jiang-hua; Yamada, Kazuya; Miyamoto, Kaoru

    2009-09-01

    We aim to investigate the effect of transforming growth factor (TGF)-beta1 on the expression of enhancer of split- and hairy-related protein-2 (SHARP-2) messenger RNA (mRNA) and its signaling pathway. In this study, several cell lines including LLC-PK1 (a porcine kidney tubular epithelial cell line), MDCK (Madin-Darby canine kidney) and CTLL-2 (cytotoxic T-lymphocyte line) were treated with recombinant human TGF-beta1, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-beta1 treatment to probe the signaling pathway. The results showed that TGF-beta1 can significantly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-beta1 stimulation. While one phosphoinositide 3-kinases (PI-3) kinase inhibitor, LY294002, completely blocked the effect of TGF-beta1 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-beta1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect. PMID:19735104

  10. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells

    PubMed Central

    Rafiq, Rather A.; Quadri, Afnan; Nazir, Lone A.; Peerzada, Kaiser; Ganai, Bashir A.; Tasduq, Sheikh A.

    2015-01-01

    Ultraviolet (UV) radiation–induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  11. Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.

    PubMed

    Bekhite, Mohamed M; Müller, Veronika; Tröger, Sebastian H; Müller, Jörg P; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2016-04-01

    The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration. PMID:26553657

  12. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells.

    PubMed

    Rafiq, Rather A; Quadri, Afnan; Nazir, Lone A; Peerzada, Kaiser; Ganai, Bashir A; Tasduq, Sheikh A

    2015-01-01

    Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  13. 5-Azacytidine regulates matrix metalloproteinase-9 expression, and the migration and invasion of human fibrosarcoma HT1080 cells via PI3-kinase and ERK1/2 pathways.

    PubMed

    Yu, Seon-Mi; Kim, Song Ja

    2016-09-01

    Abnormal methylation of promoter CpG islands is one of the hallmarks of cancer cells, and is catalyzed by DNA methyltransferases. 5-azacytidine (5-aza C), a methyltransferase inhibitor, can cause demethylation of promoter regions of diverse genes. Epigenetic processes contribute to the regulation of matrix metalloproteinase (MMP) expression. However, little is known about the mechanisms and effects of 5-aza C on the invasive and migratory capacities of human fibrosarcoma HT1080 cells. In the present study, we found that 5-aza C induces MMP-9 activity, as determined by zymography. HT1080 cell proliferation was determined following 5-aza C administration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle was examined by flow cytometry. 5-aza C treatment inhibited cell proliferation without affecting cell viability. Furthermore, 5-aza C significantly promoted migration and invasion of HT1080 cells. 5-aza C treatment enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and phosphoinositide (PI)3-kinase/Akt, and their inhibitors blocked MMP-9 activity induction, and cellular invasion and migration. Together, these findings suggest that promoter methylation may be one of the mechanisms modulating MMP-9 levels in HT1080 cells, and that 5-aza C-induced MMP-9 production is associated with the activation of ERK and PI3-kinase/Akt signaling pathways. PMID:27573026

  14. LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

    SciTech Connect

    Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E. . E-mail: tadrian@northwestern.edu

    2005-09-30

    We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.

  15. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  16. Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells.

    PubMed

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N; Hoek, Jan B; Kiyatkin, Anatoly

    2011-11-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  17. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    PubMed

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP

  18. Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

    PubMed Central

    Vinokur, Jeffrey M; Korman, Tyler P; Sawaya, Michael R; Collazo, Michael; Cascio, Duillio; Bowie, James U

    2015-01-01

    In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation. PMID:25422158

  19. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    PubMed Central

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  20. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

    PubMed Central

    Brunn, G J; Williams, J; Sabers, C; Wiederrecht, G; Lawrence, J C; Abraham, R T

    1996-01-01

    The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Images PMID:8895571

  1. A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

    SciTech Connect

    Iqbal, Mohd S.; Tsuyama, Naohiro; Obata, Masanori; Ishikawa, Hideaki

    2010-02-12

    Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21{sup Cip1} proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

  2. Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC.

    PubMed

    Wasilko, David J; Mao, Yuxin

    2016-02-01

    Intracellular bacterial pathogens use secreted effector proteins to alter host cellular processes, with the goal of subverting host defenses and allowing the infection to progress. One such pathogen, Legionella pneumophila, secretes ~300 proteins into its host to alter a number of pathways including intracellular trafficking, phosphoinositide metabolism, and cell signaling. The Legionella effector SidC was previously found to bind to PI(4)P and was responsible for the enrichment of ER proteins and ubiquitinated species on the Legionella-containing vacuoles. Through our recent work, we have discovered that SidC contains a unique N-terminal E3 ubiquitin ligase domain and a C-terminal novel PI(4)P-binding domain. Our results demonstrate that SidC serves to link two distinct cellular pathways, ubiquitin and phosphoinositide. However, how the ubiquitin ligase activity regulates host membrane trafficking events remains to be investigated. PMID:26433729

  3. Pooled Analysis of Phosphatidylinositol 3-kinase Pathway Variants and Risk of Prostate Cancer

    PubMed Central

    Koutros, Stella; Schumacher, Fredrick R.; Hayes, Richard B.; Ma, Jing; Huang, Wen-Yi; Albanes, Demetrius; Canzian, Federico; Chanock, Stephen J.; Crawford, E. David; Diver, W. Ryan; Feigelson, Heather Spencer; Giovanucci, Edward; Haiman, Christopher A.; Henderson, Brian E.; Hunter, David J.; Kaaks, Rudolf; Kolonel, Laurence N.; Kraft, Peter; Le Marchand, Loïc; Riboli, Elio; Siddiq, Afshan; Stampfer, Mier J.; Stram, Daniel O.; Thomas, Gilles; Travis, Ruth C.; Thun, Michael J.; Yeager, Meredith; Berndt, Sonja I.

    2010-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes, including cellular proliferation and intracellular trafficking and may impact prostate carcinogenesis. Thus, we explored the association between single nucleotide polymorphisms (SNPs) in PI3K genes and prostate cancer. Pooled data from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium were examined for associations between 89 SNPs in PI3K genes (PIK3C2B, PIK3AP1, PIK3C2A, PIK3CD, and PIK3R3) and prostate cancer risk in 8,309 cases and 9,286 controls. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. SNP rs7556371 in PIK3C2B was significantly associated with prostate cancer risk (ORper allele=1.08 (95% CI: 1.03, 1.14), p-trend = 0.0017) after adjustment for multiple testing (Padj=0.024). Simultaneous adjustment of rs7556371 for nearby SNPs strengthened the association (ORper allele=1.21 (95% CI: 1.09, 1.34); p-trend =0.0003). The adjusted association was stronger for men who were diagnosed before 65 years (ORper allele= 1.47 (95% CI: 1.20, 1.79), p-trend = 0.0001) or had a family history (ORper allele= 1.57 (95% CI: 1.11, 2.23), p-trend = 0.0114), and was strongest in those with both characteristics (ORper allele= 2.31 (95% CI: 1.07, 5.07), p-interaction = 0.005). Increased risks were observed among men in the top tertile of circulating insulin like growth factor-1 (IGF-1) levels (ORper allele= 1.46 (95% CI: 1.04, 2.06), p-trend=0.075). No differences were observed with disease aggressiveness (≥8/stage T3/T4/fatal). In conclusion, we observed a significant association between PIK3C2B and prostate cancer risk, especially for familial, early onset disease, which may be attributable to IGF-dependent PI3K signaling. PMID:20197460

  4. Idelalisib: Targeting the PI3 Kinase Pathway in Non-Hodgkin Lymphoma.

    PubMed

    Sujobert, Pierre; Rioufol, Catherine; Salles, Gilles A

    2016-01-01

    Based on substantial preclinical rationale, the restricted hematopoietic expression of the δ isoform of the phosphatidylinositol 3-kinase represents an attractive therapeutic target in B-cell malignancies. Its inhibition results in a direct antiproliferative effect on tumor cells as well as several modifications of their cellular microenvironment, all accounting for the potential therapeutic interest. Idelalisib, the first-in-class phosphatidylinositol 3-kinase δ-specific inhibitor, was developed in patients with B-cell lymphomas and chronic lymphocytic leukemia. Early clinical results demonstrated a potent antitumor effect across different subtypes of indolent and mantle cell lymphomas (where response duration was short). Adverse events, including transaminitis, neutropenia, pneumonitis, and diarrhea, were observed. A pivotal phase II study in patients with double refractory disease showed a 57% response rate, with response lasting for about 1 year, leading to market approval of the drug in the United States and Europe. Further developments of idelalisib combinations will contribute to delineate the position of this drug in the therapeutic strategy of indolent lymphomas. PMID:26841011

  5. Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production

    PubMed Central

    Barbera Betancourt, Ariana; Emery, Juliet L.; Recino, Asha; Wong, F. Susan; Cooke, Anne; Okkenhaug, Klaus; Wallberg, Maja

    2016-01-01

    Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3+ CD4+ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig. PMID:26783747

  6. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

    PubMed Central

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee

    2012-01-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway. PMID:23118562

  7. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway.

    PubMed

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee; Chung, Jin Woong

    2012-10-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway. PMID:23118562

  8. Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

    SciTech Connect

    Favreau, Catherine; Delbarre, Erwan; Courvalin, Jean-Claude; Buendia, Brigitte

    2008-04-01

    Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.

  9. Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: A crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

    SciTech Connect

    Bernard, Laurence; Legay, Christine; Adriaenssens, Eric; Mougel, Alexandra; Ricort, Jean-Marc . E-mail: ricort@lbpa.ens-cachan.fr

    2006-12-01

    Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110 expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.

  10. Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 while oncogenic Ras is a dominant predictor for resistance

    PubMed Central

    Ihle, NathanT.; Lemos, Robert; Wipf, Peter; Yacoub, Adly; Mitchell, Clint; Siwak, Doris; Mills, Gordon B.; Dent, Paul; Kirkpatrick, D Lynn.; Powis, Garth

    2008-01-01

    The novel phosphatidylinositol-3-kinase (PI-3-kinase) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI-3-kinase (PIK3CA) and loss of PTEN activity were sufficient but not necessary as predictors of sensitivity to the antitumor activity of the PI-3-K inhibitor PX-866 in the presence of wild type Ras, while mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI-3-kinase signaling measured by tumor phospho-Ser473-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse phase protein array (RPPA) revealed that the Ras dependent down stream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best defined downstream targets of Ras, namely Raf, RalGDS, and PI-3-kinase, showed that mutant Ras mediates resistance through its ability to utilize multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI-3-kinase inhibition may serve as an important guide for patient selection as inhibitors enter clinical trials, and for the development of rational combinations with other molecularly targeted agents. PMID:19117997

  11. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

    PubMed Central

    Logan, S K; Falasca, M; Hu, P; Schlessinger, J

    1997-01-01

    The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells. PMID:9315636

  12. Clozapine Interaction with Phosphatidyl Inositol 3-Kinase (PI3K)/Insulin Signaling Pathway in Caenorhabditis elegans

    PubMed Central

    Karmacharya, Rakesh; Sliwoski, Gregory R.; Lundy, Miriam Y.; Suckow, Raymond F.; Cohen, Bruce M.; Buttner, Edgar A.

    2012-01-01

    Clozapine has superior and unique effects as an antipsychotic agent, but the mediators of these effects are not known. We studied behavioral and developmental effects of clozapine in Caenorhabditis elegans, as a model system to identify previously undiscovered mechanisms of drug action. Clozapine induced early larval arrest, a phenotype that was also seen with the clozapine metabolite N-desmethyl clozapine but not with any other typical or atypical antipsychotic drug tested. Mutations in the insulin receptor/daf-2 and the phosphatidyl inositol 3-kinase (PI3K)/age-1 suppressed clozapine-induced larval arrest, suggesting that clozapine may activate the insulin signaling pathway. Consistent with this notion, clozapine also increased expression of an age-1::GFP reporter. Activation of the insulin signaling pathway leads to cytoplasmic localization of the fork head transcription factor FOXO/daf-16. Clozapine produced cytoplasmic localization of DAF-16::GFP in arrested L1 larvae, in contrast to stressors such as starvation or high temperature which produce nuclear localization of DAF-16::GFP in arrested L1 larvae. Clozapine also inhibited pharyngeal pumping in C. elegans, an effect that may contribute to but did not explain clozapine-induced larval arrest. Our findings demonstrate a drug-specific interaction between clozapine and the PI3K/insulin signaling pathway in C. elegans. As this pathway is conserved across species, the results may have implications for understanding the unique effects of clozapine in humans. PMID:19322168

  13. Therapeutic targeting of the phosphatidylinositol 3-kinase signaling pathway: novel targeted therapies and advances in the treatment of colorectal cancer

    PubMed Central

    Yu, Ming

    2012-01-01

    Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the USA, and more effective treatment of CRC is therefore needed. Advances in our understanding of the molecular pathogenesis of this malignancy have led to the development of novel molecule-targeted therapies. Among the most recent classes of targeted therapies being developed are inhibitors targeting the phosphatidylinositol 3-kinase (PI3K) signaling pathway. As one of the most frequently deregulated pathways in several human cancers, including CRC, aberrant PI3K signaling plays an important role in the growth, survival, motility and metabolism of cancer cells. Targeting this pathway therefore has considerable potential to lead to novel and more effective treatments for CRC. Preclinical and early clinical studies have revealed the potential efficacy of drugs that target PI3K signaling for the treatment of CRC. However, a major challenge that remains is to study these agents in phase III clinical trials to see whether these early successes translate into better patient outcomes. In this review we focus on providing an up-to-date assessment of our current understanding of PI3K signaling biology and its deregulation in the molecular pathogenesis of CRC. Advances in available agents and challenges in targeting the PI3K signaling pathway in CRC treatment will be discussed and placed in the context of the currently available therapies for CRC. PMID:22973417

  14. Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

    PubMed

    Xie, Liji; Xie, Zhixun; Huang, Li; Fan, Qing; Luo, Sisi; Huang, Jiaoling; Deng, Xianwen; Xie, Zhiqin; Zeng, Tingting; Zhang, Yanfang; Wang, Sheng

    2016-08-01

    The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, μA, μB and μNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, μA-pcAGEN, μB-pcAGEN and μNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, μA, μB and μNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., μA, μB and μNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway. PMID:27233800

  15. p110δ PI3 kinase pathway: emerging roles in cancer

    PubMed Central

    Tzenaki, Niki; Papakonstanti, Evangelia A.

    2012-01-01

    Class IA PI3Ks consists of three isoforms of the p110 catalytic subunit designated p110α, p110β, and p110δ which are encoded by three separate genes. Gain-of-function mutations on PIK3CA gene encoding for p110α isoform have been detected in a wide variety of human cancers whereas no somatic mutations of genes encoding for p110β or p110δ have been reported. Unlike p110α and p110β which are ubiquitously expressed, p110δ is highly enriched in leukocytes and thus the p110δ PI3K pathway has attracted more attention for its involvement in immune disorders. However, findings have been accumulated showing that the p110δ PI3K plays a seminal role in the development and progression of some hematologic malignancies. A wealth of knowledge has come from studies showing the central role of p110δ PI3K in B-cell functions and B-cell malignancies. Further data have documented that wild-type p110δ becomes oncogenic when overexpressed in cell culture models and that p110δ is the predominant isoform expressed in some human solid tumor cells playing a prominent role in these cells. Genetic inactivation of p110δ in mice models and highly-selective inhibitors of p110δ have demonstrated an important role of this isoform in differentiation, growth, survival, motility, and morphology with the inositol phosphatase PTEN to play a critical role in p110δ signaling. In this review, we summarize our understanding of the p110δ PI3K signaling pathway in hematopoietic cells and malignancies, we highlight the evidence showing the oncogenic potential of p110δ in cells of non-hematopoietic origin and we discuss perspectives for potential novel roles of p110δ PI3K in cancer. PMID:23459844

  16. A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Koushik, Amrita B.; Welter, Brenda H.; Rock, Michelle L.

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. PMID:24442890

  17. Isoorientin induces Nrf2 pathway-driven antioxidant response through phosphatidylinositol 3-kinase signaling.

    PubMed

    Lim, Ju Hee; Park, Hae-Suk; Choi, Jung-Kap; Lee, Ik-Soo; Choi, Hyun Jin

    2007-12-01

    Because oxidative stress is involved in the pathogenesis of various chronic diseases and the aging process, antioxidants that can increase the intrinsic antioxidant potency are proposed as desirable therapeutic agents to counteract oxidative stress-related diseases. NF-E2-related factor-2 (Nrf2) is a transcription factor that regulates important antioxidant and phase II detoxification genes, and therefore, the molecule that regulates nuclear translocation of Nrf2 and the induction of antioxidative proteins is thought to be a promising candidate as a cytoprotective agent for oxidative stress. In the present study, we show that isoorientin (luteolin 6-C-beta-D-glucoside) obtained from the leaves of Sasa borealis upregulates and activates Nrf2, and has protective ability against oxidative damage caused by reactive oxygen intermediates in HepG2 cells. Isoorientin induces increase in the level of antioxidant enzyme proteins, especially NQO1, and the cytoprotective and antioxidative effects of isoorientin are PI3K/Akt pathway-dependent. Together with direct radical scavenging activity, the novel effect of isoorientin on the regulation of antioxidative gene expression provides attractive strategy to prevent diseases associated with oxidative stress and attenuate the progress of the diseases. PMID:18254247

  18. PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma.

    PubMed

    Kanteti, Rajani; Riehm, Jacob J; Dhanasingh, Immanuel; Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Bolot; Kindler, Hedy L; Salgia, Ravi

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent. PMID:27623107

  19. Phosphatidylinositol 3-kinase in myogenesis.

    PubMed

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc. PMID:21235885

  20. The Phosphatidylinositol 3-Kinase/Akt Pathway Negatively Regulates Nod2-Mediated NF-kB Pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways including NF-kB pathway. Here, we show that Nod2 agonist MDP induces Ak...

  1. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  2. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  3. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells.

    PubMed

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A

    2016-04-01

    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes. PMID:26786777

  4. Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

    PubMed Central

    Kalachova, Tetiana; Kravets, Volodymyr; Zachowski, Alain; Ruelland, Eric

    2015-01-01

    “Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones. PMID:26039482

  5. Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway.

    PubMed

    Kaliman, P; Canicio, J; Testar, X; Palacín, M; Zorzano, A

    1999-06-18

    Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB. PMID:10364173

  6. Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase

    PubMed Central

    Pinson, Jo-Anne; Schmidt-Kittler, Oleg; Frazzetto, Mark; Zheng, Zhaohua; Jennings, Ian G.; Kinzler, Kenneth W.; Vogelstein, Bert; Chalmers, David K.; Thompson, Philip E.

    2012-01-01

    The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity. PMID:23997244

  7. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways.

    PubMed

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P; Taub, Dennis D

    2014-12-20

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  8. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    PubMed Central

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  9. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  10. Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway.

    PubMed

    Mao, Mao; Sudhahar, Varadarajan; Ansenberger-Fricano, Kristine; Fernandes, Denise C; Tanaka, Leonardo Y; Fukai, Tohru; Laurindo, Francisco R M; Mason, Ronald P; Vasquez-Vivar, Jeannette; Minshall, Richard D; Stadler, Krisztian; Bonini, Marcelo G

    2012-01-15

    Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1-50nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP(3), probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses. PMID:22037515

  11. Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

    PubMed Central

    Mao, Mao; Sudhahar, Varadarajan; Ansenberger-Fricano, Kristine; Fernandes, Denise C.; Tanaka, Leonardo Y.; Fukai, Tohru; Laurindo, Francisco R.M.; Mason, Ronald P.; Vasquez-Vivar, Jeannette; Minshall, Richard D.; Stadler, Krisztian; Bonini, Marcelo G.

    2012-01-01

    Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1–50 nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP3, probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses. PMID:22037515

  12. Upregulation of heme oxygenase-1 by Epigallocatechin-3-gallate via the phosphatidylinositol 3-kinase/Akt and ERK pathways.

    PubMed

    Wu, C C; Hsu, M C; Hsieh, C W; Lin, J B; Lai, P H; Wung, B S

    2006-05-15

    Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various phytochemicals and we examined the ability of Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, to upregulate HO-1 expression in endothelial cells (ECs). We demonstrate that EGCG induces HO-1 expression in a concentration- and time-dependent manner. Furthermore, EGCG-mediated HO-1 induction was abrogated in the presence of actinomycin D and cycloheximide, indicating that this upregulation of HO-1 occurred at the transcriptional level. EGCG also upregulates Nrf2 levels in nuclear extracts and increases ARE-luciferase activity. Furthermore, EGCG is the most potent inducer of HO-1 expression of the different green tea constituents that we analyzed, but had no detectable cytotoxic effects over the 25-100 microM dosage range. The inhibition of intracellular ROS production by N-acetylcysteine (NAC), glutathione (GSH), superoxide dismutase (SOD), catalase and the mitochondrial complex I inhibitor, rotenone, results in a decrease in EGCG-dependent HO-1 expression. In addition, we determined that tyrosine kinase is involved in EGCG induction of HO-1 as this is abrogated by genistein. ECs treated with EGCG exhibit activation of Akt and ERK1/2. In addition, pharmacological inhibitors of phosphatidylinositol 3-kinase and MEK1/2, which are upstream of Akt and ERK1/2, respectively, attenuate EGCG-induced HO-1 expression. On the other hand, pretreatment of these cells with EGCG exerts significant cytoprotective effects against H2O2, suggesting that the induction of HO-1 is an important component in the protection against oxidative stress. Hence, EGCG is a novel phytochemical inducer of HO-1 expression and we further identify the principal underlying mechanisms involved in this process. PMID:16378625

  13. The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326).

    PubMed

    Heffron, Timothy P; Heald, Robert A; Ndubaku, Chudi; Wei, BinQing; Augistin, Martin; Do, Steven; Edgar, Kyle; Eigenbrot, Charles; Friedman, Lori; Gancia, Emanuela; Jackson, Philip S; Jones, Graham; Kolesnikov, Aleksander; Lee, Leslie B; Lesnick, John D; Lewis, Cristina; McLean, Neville; Mörtl, Mario; Nonomiya, Jim; Pang, Jodie; Price, Steve; Prior, Wei Wei; Salphati, Laurent; Sideris, Steve; Staben, Steven T; Steinbacher, Stefan; Tsui, Vickie; Wallin, Jeffrey; Sampath, Deepak; Olivero, Alan G

    2016-02-11

    Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kβ relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4). PMID:26741947

  14. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

    SciTech Connect

    Li, Ying; Wang, Jianwei; Gu, Tieguang; Yamahara, Johji; Li, Yuhao

    2014-06-01

    Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in

  15. Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival.

    PubMed

    Costa, Adriana Fraga; Gomes, Sara Zago; Lorenzon-Ojea, Aline R; Martucci, Mariane; Faria, Miriam Rubio; Pinto, Décio Dos Santos; Oliveira, Sergio F; Ietta, Francesca; Paulesu, Luana; Bevilacqua, Estela

    2016-05-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation. PMID:27208405

  16. Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase signaling pathways.

    PubMed

    Fitsialos, Giorgos; Chassot, Anne-Amandine; Turchi, Laurent; Dayem, Manal A; LeBrigand, Kevin; Moreilhon, Chimène; Meneguzzi, Guerrino; Buscà, Roser; Mari, Bernard; Barbry, Pascal; Ponzio, Gilles

    2007-05-18

    Covering denuded dermal surfaces after injury requires migration, proliferation, and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38(MAPK) and phosphatidylinositol 3-kinase (PI3K), demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. p38(MAPK) inhibition only delays "healing," probably in line with the control of genes involved in the propagation of injury-initiated signaling. In contrast, PI3K inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF, and ETS1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38(MAPK) and negative ones triggered by PI3K. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids. PMID:17363378

  17. Extrapancreatic roles of glimepiride on osteoblasts from rat manibular bone in vitro: Regulation of cytodifferentiation through PI3-kinases/Akt signalling pathway.

    PubMed

    Ma, Pan; Xiong, Wei; Liu, Hongchen; Ma, Junli; Gu, Bin; Wu, Xia

    2011-04-01

    Glimepiride, a third-generation sulfonylurea, has also been reported to have extrapancreatic functions including activation of PI3-kinase (PI3K) and Akt in rat adipocytes, skeletal muscle and endothelial cells. It is tempting to speculate that glimepiride would improve bone-implant contact in diabetic patients by mediating the activity of GLUT1 and 3 via the PI3K/Akt pathway. In this study, we investigated the effects of glimepiride on rat mandible osteoblasts cultured under two different levels of glucose. Cell proliferation was determined by the MTT assay. The supernatant was used to measure alkaline phosphatase (ALP) activity. Glucose uptake was determined by measuring the rate of 2-deoxy-d-glucose (2-DG) uptake. Western blotting was performed used to determine collagen I and PI3K/Akt expression. RT-PCR was performed used to determine osteocalcin (OCN) mRNA expression. We found that hyperglycemia down-regulated proliferation, ALP activity, OCN mRNA and GLUT3 protein expression in rat osteoblasts, and upregulated collagen I and GLUT1 protein expressions. Glimepiride enhanced the proliferation, ALP activity and OCN mRNA levels, and upregulated collagen I and GLUT1 and 3 protein expressions of rat osteoblasts at two different glucose concentrations. This study also provides the first evidence that glimepiride stimulates the phosphorylation of PI3K/Akt in osteoblasts and ameliorated the damage caused by high concentrations of glucose through the PI3K/Akt pathway. PMID:21055727

  18. Rosiglitazone-induced myocardial protection against ischaemia-reperfusion injury is mediated via a phosphatidylinositol 3-kinase/Akt-dependent pathway.

    PubMed

    Zhang, Xue-Jiao; Xiong, Zi-Bo; Tang, An-Li; Ma, Hong; Ma, Yue-Dong; Wu, Jing-Guo; Dong, Yu-Gang

    2010-02-01

    1. Rosiglitazone is widely used in the treatment of Type 2 diabetes. However, in recent years it has become evident that the therapeutic effects of peroxisome proliferator-activated receptor gamma ligands reach far beyond their use as insulin sensitizers. Recently, the ability of rosiglitazone pretreatment to induce cardioprotection following ischaemia-reperfusion (I/R) has been well documented; however, the protective mechanisms have not been elucidated. In the present study, examined the role of the phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway in rosiglitazone cardioprotection following I/R injury. 2. Mice were pretreated with 3 mg/kg per day rosiglitazone for 14 days before hearts were subjected to ischaemia (30 min) and reperfusion (2 h). Wortmannin (1.4 mg/kg, i.p.), an inhibitor of PI3-K, was administered 10 min prior to myocardial I/R. Then, activation of the PI3-K/Akt/glycogen synthase kinase (GSK)-3alpha signalling pathway was examined. The effects of PI3-K inhibition on rosiglitazone-induced cardioprotection were also evaluated. 3. Compared with control rats, the ratio of infarct size to ischaemic area (area at risk) and the occurrence of sustained ventricular fibrillation in rosiglitazone-pretreated rats was significantly reduced (P < 0.05). Rosiglitazone pretreatment attenuated cardiac apoptosis, as assessed by ELISA to determine cardiomyocyte DNA fragmentation. Rosiglitazone pretreatment significantly increased levels of phosphorylated (p-) Akt and p-GSK-3alpha in the rat myocardium. Pharmacological inhibition of PI3-K by wortmannin markedly abolished the cardioprotection induced by rosiglitazone. 4. These results indicate that rosiglitazone-induced cardioprotection in I/R injury is mediated via a PI3-K/Akt/GSK-3alpha-dependent pathway. The data also suggest that modulation of PI3-K/Akt/GSK-3alpha-dependent signalling pathways may be a viable strategy to reduce myocardial I/R injury. PMID:19566839

  19. Neurotoxicity of developmental hypothyroxinemia and hypothyroidism in rats: Impairments of long-term potentiation are mediated by phosphatidylinositol 3-kinase signaling pathway

    SciTech Connect

    Wang, Yi; Wei, Wei; Wang, Yuan; Dong, Jing; Song, Binbin; Min, Hui; Teng, Weiping; Chen, Jie

    2013-09-01

    Neurotoxicity of iodine deficiency-induced hypothyroidism during developmental period results in serious impairments of brain function, such as learning and memory. These impairments are largely irreversible, and the underlying mechanisms remain unclear. In addition to hypothyroidism, iodine deficiency may cause hypothyroxinemia, a relatively subtle form of thyroid hormone deficiency. Neurotoxicity of developmental hypothyroxinemia also potentially impairs learning and memory. However, more direct evidence of the associations between developmental hypothyroxinemia and impairments of learning and memory should be provided, and the underlying mechanisms remain to be elucidated. Thus, in the present study, we investigated the effects of developmental hypothyroxinemia and hypothyroidism on long-term potentiation (LTP), a widely accepted cellular model of learning and memory, in the hippocampal CA1 region. The activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway – a pathway closely associated with synaptic plasticity and learning and memory – was also investigated. Wistar rats were treated with iodine deficient diet or methimazole (MMZ) to induce developmental hypothyroxinemia or hypothyroidism. The results showed that developmental hypothyroxinemia caused by mild iodine deficiency and developmental hypothyroidism caused by severe iodine deficiency or MMZ significantly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Decreased activation of the PI3K signaling pathway was also observed in rats subjected to developmental hypothyroxinemia or hypothyroidism. Our results may support the hypothesis that neurotoxicity of both developmental hypothyroxinemia and hypothyroidism causes damages to learning and memory. Our results also suggest that decreased activation of the PI3K signaling pathway may contribute to impairments of LTP caused by neurotoxicity of both developmental hypothyroxinemia and

  20. Osthole relaxes pulmonary arteries through endothelial phosphatidylinositol 3-kinase/Akt-eNOS-NO signaling pathway in rats.

    PubMed

    Yao, Li; Lu, Ping; Li, Yumei; Yang, Lijing; Feng, Hongxuan; Huang, Yong; Zhang, Dandan; Chen, Jianguo; Zhu, Daling

    2013-01-15

    Pulmonary arterial hypertension is a life-threatening disease lacking effective therapies. Osthole is a natural coumarin compound isolated from Angelica pubescens Maxim., which possesses hypotensive effect. Although its effects on isolated thoracic aorta (systemic circulating system) are clarified, it remains unclear whether Osthole relaxes isolated pulmonary arteries (PAs) (pulmonary circulating system). The aim of this study was to investigate the effects of Osthole on isolated PAs and the underlying mechanisms. We examined PA relaxation induced by Osthole in isolated human and rat PA rings with force-electricity transducers, the expression and activity of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt) with western blot, and nitric oxide (NO) production using DAF-FM DA fluorescent indicator. The results showed that Osthole elicited a dose-dependent vasorelaxation activity with phenylephrine-precontracted human and rat PA rings, which can be diminished by endothelium denudation and inhibition of eNOS, while having no effect on rat mesenteric arteries. Osthole increased NO release as well as activation of Akt and eNOS, indicated with increased phosphorylations of Akt at Ser-473 and eNOS at Ser-1177 in endothelial cells. PI3K inhibitor LY294002 also blocked Osthole induced vasodilation. In summary, dilative effect of Osthole was dependent on endothelial integrity and NO production, and was mediated by endothelial PI3K/Akt-eNOS-NO pathway. These may provide a new pulmonary vasodilator for the therapy of pulmonary arterial hypertension. PMID:23220709

  1. The modulation of vascular ATP-sensitive K+ channel function via the phosphatidylinositol 3-kinase-Akt pathway activated by phenylephrine.

    PubMed

    Haba, Masanori; Hatakeyama, Noboru; Kinoshita, Hiroyuki; Teramae, Hiroki; Azma, Toshiharu; Hatano, Yoshio; Matsuda, Naoyuki

    2010-08-01

    The present study examined the modulator role of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway activated by the alpha-1 adrenoceptor agonist phenylephrine in ATP-sensitive K(+) channel function in intact vascular smooth muscle. We evaluated the ATP-sensitive K(+) channel function and the activity of the PI3K-Akt pathway in the rat thoracic aorta without endothelium. The PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (10(-5) M) augmented relaxation in response to the ATP-sensitive K(+) channel opener levcromakalim (10(-8) to 3 x 10(-6) M) in aortic rings contracted with phenylephrine (3 x 10(-7) M) but not with 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619; 3 x 10(-8) M), although those agents induced similar contraction. ATP-sensitive K(+) channel currents induced by levcromakalim (10(-6) M) in the presence of phenylephrine (3 x 10(-7) M) were enhanced by the nonselective alpha-adrenoceptor antagonist phentolamine (10(-7) M) and LY294002 (10(-5) M). Levels of the regulatory subunits of PI3K p85-alpha and p55-gamma increased in the membrane fraction from aortas without endothelium treated with phenylephrine (3 x 10(-7) M) but not with U46619 (3 x 10(-8) M). Phenylephrine simultaneously augmented Akt phosphorylation at Ser473 and Thr308. Therefore, activation of the PI3K-Akt pathway seems to play a role in the impairment of ATP-sensitive K(+) channel function in vascular smooth muscle exposed to alpha-1 adrenergic stimuli. PMID:20519555

  2. Inhibition of gap junctional Intercellular communication in WB-F344 rat liver epithelial cells by triphenyltin chloride through MAPK and PI3-kinase pathways

    PubMed Central

    2010-01-01

    Background Organotin compounds (OTCs) have been widely used as stabilizers in the production of plastic, agricultural pesticides, antifoulant plaints and wood preservation. The toxicity of triphenyltin (TPT) compounds was known for their embryotoxic, neurotoxic, genotoxic and immunotoxic effects in mammals. The carcinogenicity of TPT was not well understood and few studies had discussed the effects of OTCs on gap junctional intercellular communication (GJIC) of cells. Method In the present study, the effects of triphenyltin chloride (TPTC) on GJIC in WB-F344 rat liver epithelial cells were evaluated, using the scrape-loading dye transfer technique. Results TPTC inhibited GJIC after a 30-min exposure in a concentration- and time-dependent manner. Pre-incubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 and PI3K inhibitor LY294002 decreased substantially the inhibition of GJIC by TPTC. After WB-F344 cells were exposed to TPTC, phosphorylation of Cx43 increased as seen in Western blot analysis. Conclusions These results show that TPTC inhibits GJIC in WB-F344 rat liver epithelial cells by altering the Cx43 protein expression through both MAPK and PI3-kinase pathways. PMID:20591183

  3. Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

    PubMed Central

    Liu, Bin; Deng, Yi-Shu; Zhan, Dong; Chen, Yuan-Li; He, Ying; Liu, Jing; Zhang, Zong-Ji; Sun, Jun; Lu, Di

    2012-01-01

    Background Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells. Methodology/Principal Findings Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1. Conclusion and Implications This investigation

  4. Blueberry Phytochemicals Inhibit Growth and Metastatic Potential of MDA-MB-231 Breast Cancer Cells Through Modulation of the Phosphatidylinositol 3-Kinase Pathway

    PubMed Central

    Adams, Lynn S.; Phung, Sheryl; Yee, Natalie; Seeram, Navindra P.; Li, Liya; Chen, Shiuan

    2010-01-01

    Dietary phytochemicals are known to exhibit a variety of anti-carcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937 and MDA-MB-231 cells with no effect on the non-tumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound healing assays and migration through a PET membrane. Blueberry treatment decreased the activity of matrix metalloproteinase 9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa-B (NFκB) activation in MDA-MB-231 cells where protein kinase C (PKC) and extracellular regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry treated mice, where apoptosis (caspase-3 expression) was increased compared to controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFκB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFκB pathway. PMID:20388778

  5. Protective role of PI3-kinase-Akt-eNOS signalling pathway in intestinal injury associated with splanchnic artery occlusion shock

    PubMed Central

    Roviezzo, F; Cuzzocrea, S; Di Lorenzo, A; Brancaleone, V; Mazzon, E; Di Paola, R; Bucci, M; Cirino, G

    2007-01-01

    Background and purpose: Endothelial NO synthase (eNOS) is a dynamic enzyme tightly controlled by co- and post-translational lipid modifications, phosphorylation and regulated by protein-protein interactions. Here we have pharmacologically modulated the activation of eNOS, at different post-translational levels, to assess the role of eNOS-derived NO and of these regulatory mechanisms in intestinal injury associated with splanchnic artery occlusion (SAO) shock. Experimental approach: SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk for 45 min followed by 30 min of reperfusion. During ischemia, 15 min prior to reperfusion, mice were given geldanamycin, an inhibitor of hsp90 recruitment to eNOS, or LY-294002 an inhibitor of phosphatidylinositol 3-kinase (PI3K), an enzyme that initiates Akt–catalysed phosphorylation of eNOS on Ser1179. After 30 min of reperfusion, samples of ileum were taken for histological examination or for biochemical studies. Key results: Either LY-294002 or geldanamycin reversed the increased activation of eNOS and Akt observed following SAO shock. These molecular effects were mirrored in vivo by an exacerbation of the intestinal damage. Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity, and with an increased expression of the adhesion proteins: ICAM-I, VCAM, P-selectin and E-selectin. Conclusions and implications: Overall these results suggest that activation of the Akt pathway in ischemic regions of reperfused ileum is a protective event, triggered in order to protect the intestinal tissue from damage induced by ischaemia/reperfusion through a fine tuning of the endothelial NO pathway. PMID:17450173

  6. The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway.

    PubMed

    Wang, Dongdong; Saga, Yasushi; Sato, Naoto; Nakamura, Toshikazu; Takikawa, Osamu; Mizukami, Hiroaki; Matsubara, Shigeki; Fujiwara, Hiroyuki

    2016-06-01

    Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

  7. The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

    PubMed Central

    WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

    2016-01-01

    Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

  8. Expression of a phosphorylated p130Cas substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

    PubMed Central

    Soni, Shefali; Lin, Bor-Tyh; August, Avery; Nicholson, Robert I.; Kirsch, Kathrin H.

    2009-01-01

    Elevated expression of p130Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM-R cells, which were established by long-term exposure of estrogen (E2)-dependent MCF-7 cells to tamoxifen, displayed elevated levels of total and activated p130Cas. Here we have investigated the effects of p130Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130Cas, a phosphorylated substrate domain of p130Cas, that acts as a dominant-negative (DN) p130Cas molecule by blocking signal transduction downstream of the p130Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130Cas signaling/expression induced morphological changes, which were consistent with a more epithelial-like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3-kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti-apoptotic protein Bcl-2. Importantly, these changes re-sensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers. PMID:19330798

  9. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

  10. Siglec-15 regulates osteoclast differentiation by modulating RANKL-induced phosphatidylinositol 3-kinase/Akt and Erk pathways in association with signaling Adaptor DAP12.

    PubMed

    Kameda, Yusuke; Takahata, Masahiko; Komatsu, Miki; Mikuni, Shintaro; Hatakeyama, Shigetsugu; Shimizu, Tomohiro; Angata, Takashi; Kinjo, Masataka; Minami, Akio; Iwasaki, Norimasa

    2013-12-01

    Siglecs are a family of sialic acid-binding immunoglobulin-like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec-15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor κB ligand (RANKL) signaling in association with DNAX-activating protein 12 kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine-based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec-15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec-15-deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec-15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL-induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt and Erk pathways were impaired in Siglec-15-deficient cells. Retroviral transduction of Siglec-15-null osteoclast precursors with wild-type Siglec-15 or mutant Siglec-15 revealed that the association of Siglec-15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec-15-deficient mice, indicating that there is a compensatory mechanism for Siglec-15-mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast-associated receptor (OSCAR)/Fc receptor common γ (FcRγ) signaling, an alternative ITAM-mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec-15-deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec-15-deficient cells when cultured on bone slices, indicating that Siglec-15-mediated signaling can be compensated for by signaling activated by type II collagen and other bone

  11. Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity.

    PubMed

    Liu, Su-Yang; Joseph, Nancy M; Ravindranathan, Ajay; Stohr, Bradley A; Greenland, Nancy Y; Vohra, Poonam; Hosfield, Elizabeth; Yeh, Iwei; Talevich, Eric; Onodera, Courtney; Van Ziffle, Jessica A; Grenert, James P; Bastian, Boris C; Chen, Yunn-Yi; Krings, Gregor

    2016-09-01

    Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with

  12. Aspergillus fumigatus-induced Interleukin-8 Synthesis by Respiratory Epithelial Cells Is Controlled by the Phosphatidylinositol 3-Kinase, p38 MAPK, and ERK1/2 Pathways and Not by the Toll-like Receptor-MyD88 Pathway*

    PubMed Central

    Balloy, Viviane; Sallenave, Jean-Michel; Wu, Yongzheng; Touqui, Lhousseine; Latgé, Jean-Paul; Si-Tahar, Mustapha; Chignard, Michel

    2008-01-01

    Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-κB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-κB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis. PMID:18703508

  13. Aspergillus fumigatus-induced interleukin-8 synthesis by respiratory epithelial cells is controlled by the phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2 pathways and not by the toll-like receptor-MyD88 pathway.

    PubMed

    Balloy, Viviane; Sallenave, Jean-Michel; Wu, Yongzheng; Touqui, Lhousseine; Latgé, Jean-Paul; Si-Tahar, Mustapha; Chignard, Michel

    2008-11-01

    Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-kappaB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-kappaB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis. PMID:18703508

  14. G-protein coupled receptor 34 activates Erk and phosphatidylinositol 3-kinase/Akt pathways and functions as alternative pathway to mediate p185Bcr-Abl-induced transformation and leukemogenesis.

    PubMed

    Zuo, Bo; Li, Mei; Liu, Yulan; Li, Kun; Ma, Shuyun; Cui, Meihua; Qin, Yazhen; Zhu, Honghu; Pan, Xiuying; Guo, Jingzhu; Dai, Zonghan; Yu, Weidong

    2015-07-01

    Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis. PMID:25363403

  15. Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

    SciTech Connect

    Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya; Hayashi, Norio; Takehara, Tetsuo

    2011-08-19

    Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

  16. Daily Exposure to Di(2-ethylhexyl) Phthalate Alters Estrous Cyclicity and Accelerates Primordial Follicle Recruitment Potentially Via Dysregulation of the Phosphatidylinositol 3-Kinase Signaling Pathway in Adult Mice1

    PubMed Central

    Hannon, Patrick R.; Peretz, Jackye; Flaws, Jodi A.

    2014-01-01

    ABSTRACT Humans are exposed daily to di(2-ethylhexyl) phthalate (DEHP), a plasticizer found in many consumer, medical, and building products containing polyvinyl chloride. Large doses of DEHP disrupt normal ovarian function; however, the effects of DEHP at environmentally relevant levels, the effects of DEHP on folliculogenesis, and the mechanisms by which DEHP disrupts ovarian function are unclear. The present study tested the hypothesis that relatively low levels of DEHP disrupt estrous cyclicity as well as accelerate primordial follicle recruitment by dysregulating phosphatidylinositol 3-kinase (PI3K) signaling. Adult CD-1 mice were orally dosed with DEHP (20 μg/kg/day–750 mg/kg/day) daily for 10 and 30 days. Following dosing, the effects on estrous cyclicity were examined, and follicle numbers were histologically quantified. Further, the ovarian mRNA and protein levels of PI3K signaling factors that are associated with early folliculogenesis were quantified. The data indicate that 10- and 30-day exposure to DEHP prolonged the duration of estrus and accelerated primordial follicle recruitment. Specifically, DEHP exposure decreased the percentage of primordial follicles and increased the percentage of primary follicles counted following 10-day exposure and increased the percentage of primary follicles counted following 30-day exposure. DEHP exposure, at doses that accelerate folliculogenesis, increased the levels of 3-phosphoinositide-dependent protein kinase-1, mammalian target of rapamycin complex 1, and protein kinase B and decreased the levels of phosphatase and tensin homolog, potentially driving PI3K signaling. Collectively, relatively low levels of DEHP disrupt estrous cyclicity and accelerate primordial follicle recruitment potentially via a mechanism involving dysregulation of PI3K signaling. PMID:24804967

  17. DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

    PubMed

    Yu, Seon-Mi; Kim, Song-Ja

    2015-10-01

    The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells. PMID:26238650

  18. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    SciTech Connect

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-07-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

  19. PI3 kinase regulation of skeletal muscle hypertrophy and atrophy.

    PubMed

    Glass, David J

    2010-01-01

    Activation of the PI3 kinase pathway can induce skeletal muscle hypertrophy, defined as an increase in skeletal muscle mass. In mammals, skeletal muscle hypertrophy occurs as a result of an increase in the size, as opposed to the number, of pre-existing skeletal muscle fibers. This pathway's effects on skeletal muscle have been implicated most prominently downstream of Insulin-like growth factor 1 signaling. IGF-1's pro-hypertrophy activity comes predominantly through its ability to activate the Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Akt is a serine-threonine protein kinase that can induce protein synthesis and block the transcriptional upregulation of key mediators of skeletal muscle atrophy, the E3 ubiquitin ligases MuRF1 and MAFbx (also called Atrogin-1), by phosphorylating and thereby inhibiting the nuclear translocation of the FOXO (also called "forkhead") family of transcription factors. Once phosphorylated by Akt, the FOXOs are excluded from the nucleus, and upregulation of MuRF1 and MAFbx is blocked. MuRF1 and MAFbx mediate atrophy by ubiquitinating particular protein substrates, causing them to undergo degradation by the proteasome. MuRF1's substrates include several components of the sarcomeric thick filament, including Myosin Heavy Chain (MyHC). Thus, by blocking MuRF1 activation, IGF-1 helps prevent the breakdown of the thick filament under atrophy conditions.IGF1/PI3K/Akt signaling also can dominantly inhibit the effects of a secreted protein called "myostatin," which is a member of the TGFβ family of proteins. Deletion or inhibition of myostatin causes an increase in skeletal muscle size, because myostatin acts both to inhibit myoblast differentiation and to block the Akt pathway. Thus by blocking myostatin, PI3K/Akt activation stimulates differentiation and protein synthesis by this distinct mechanism. Myostatin induces the phosphorylation and activation of the transcription factors of Smad2 and Smad3, downstream of the Act

  20. Regulation of Golgi function via phosphoinositide lipids.

    PubMed

    Mayinger, Peter

    2009-09-01

    Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved from yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. PMID:19508852

  1. Regulation of Golgi function via phosphoinositide lipids

    PubMed Central

    Mayinger, Peter

    2009-01-01

    Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved form yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. PMID:19508852

  2. Phosphoinositides: Key modulators of energy metabolism☆

    PubMed Central

    Bridges, Dave; Saltiel, Alan R.

    2014-01-01

    Phosphoinositides are key players in many trafficking and signaling pathways. Recent advances regarding the synthesis, location and functions of these lipids have dramatically improved our understanding of how and when these lipids are generated and what their roles are in animal physiology. In particular, phosphoinositides play a central role in insulin signaling, and manipulation of PtdIns(3,4,5)P3 levels in particular, may be an important potential therapeutic target for the alleviation of insulin resistance associated with obesity and the metabolic syndrome. In this article we review the metabolism, regulation and functional roles of phosphoinositides in insulin signaling and the regulation of energy metabolism. This article is part of a Special Issue entitled Phosphoinositides. PMID:25463477

  3. Phosphoinositides regulate ion channels

    PubMed Central

    Hille, Bertil; Dickson, Eamonn J.; Kruse, Martin; Vivas, Oscar; Suh, Byung-Chang

    2014-01-01

    Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. PMID:25241941

  4. Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway.

    PubMed Central

    Pal, M; Ishigaki, Y; Nagy, E; Maquat, L E

    2001-01-01

    Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. PMID:11214180

  5. Phosphoinositide signaling.

    PubMed

    Boss, Wendy F; Im, Yang Ju

    2012-01-01

    "All things flow and change…even in the stillest matter there is unseen flux and movement." Attributed to Heraclitus (530-470 BC), from The Story of Philosophy by Will Durant. Heraclitus, a Greek philosopher, was thinking on a much larger scale than molecular signaling; however, his visionary comments are an important reminder for those studying signaling today. Even in unstimulated cells, signaling pathways are in constant metabolic flux and provide basal signals that travel throughout the organism. In addition, negatively charged phospholipids, such as the polyphosphorylated inositol phospholipids, provide a circuit board of on/off switches for attracting or repelling proteins that define the membranes of the cell. This template of charged phospholipids is sensitive to discrete changes and metabolic fluxes-e.g., in pH and cations-which contribute to the oscillating signals in the cell. The inherent complexities of a constantly fluctuating system make understanding how plants integrate and process signals challenging. In this review we discuss one aspect of lipid signaling: the inositol family of negatively charged phospholipids and their functions as molecular sensors and regulators of metabolic flux in plants. PMID:22404474

  6. Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin

    PubMed Central

    Majeed, R; Hamid, A; Sangwan, P L; Chinthakindi, P K; Koul, S; Rayees, S; Singh, G; Mondhe, D M; Mintoo, M J; Singh, S K; Rath, S K; Saxena, A K

    2014-01-01

    Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich

  7. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  8. In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways.

    PubMed

    Tan, Bee K; Chen, Jing; Brown, James; Adya, Raghu; Ramanjaneya, Manjunath; Menon, Vinod; Bailey, Clifford J; Lehnert, Hendrik; Randeva, Harpal S

    2009-08-01

    Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P < 0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10(-9) M, returning to baseline at leptin 10(-7) M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P < 0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10(-7) M (P < 0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin receptor gene was knocked down using small interfering RNA, leptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin. PMID:19389835

  9. P38 AND EGF RECEPTOR KINASE-MEDIATED ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IS REQUIRED FOR ZN2+INDUCED CYCLOOXYGENASE-2 EXPRESSION

    EPA Science Inventory

    Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus- and cell type-specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction...

  10. Growth-stimulatory activity of TIMP-2 is mediated through c-Src activation followed by activation of FAK, PI3-kinase/AKT, and ERK1/2 independent of MMP inhibition in lung adenocarcinoma cells

    PubMed Central

    Kim, Tae Hyun; Lee, Ju-Seog; Lee, Seung-Taek; Lee, Seo-Jin

    2015-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) control extracellular matrix (ECM) homeostasis by inhibiting the activity of matrix metalloproteinases (MMPs), which are associated with ECM turnover. Recent studies have revealed that TIMPs are implicated in tumorigenesis in both MMP-dependent and MMP-independent manners. We examined a mechanism by which TIMP-2 stimulated lung adenocarcinoma cell proliferation, independent of MMP inhibition. The stimulation of growth by TIMP-2 in A549 cells required c-Src kinase activation. c-Src kinase activity, induced by TIMP-2, concomitantly increased FAK, phosphoinositide 3-kinase (PI3-kinase)/AKT, and ERK1/2 activation. Selective knockdown of integrin α3β1, known as a TIMP-2 receptor, did not significantly change TIMP-2 growth promoting activity. Furthermore, we showed that high TIMP-2 expression in lung adenocarcinomas is associated with a worse prognosis from multiple cohorts, especially for stage I lung adenocarcinoma. Through integrated analysis of The Cancer Genome Atlas data, TIMP-2 expression was significantly associated with the alteration of driving genes, c-Src activation, and PI3-kinase/AKT pathway activation. Taken together, our results demonstrate that TIMP-2 stimulates lung adenocarcinoma cell proliferation through c-Src, FAK, PI3-kinase/AKT, and ERK1/2 pathway activation in an MMP-independent manner. PMID:26556867

  11. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  12. E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

    SciTech Connect

    Contreras-Paredes, Adriana

    2009-01-05

    Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.

  13. IL-15 renders conventional lymphocytes resistant to suppressive functions of regulatory T cells through activation of the phosphatidylinositol 3-kinase pathway.

    PubMed

    Ben Ahmed, Mélika; Belhadj Hmida, Nadia; Moes, Nicolette; Buyse, Sophie; Abdeladhim, Maha; Louzir, Hechmi; Cerf-Bensussan, Nadine

    2009-06-01

    IL-15 drives chronic inflammation in several human diseases. We have recently shown that IL-15 inhibits the immunosuppressive effects of TGF-beta through blockage of the Smad3-signaling pathway. Data pointing to reciprocal interactions between TGF-beta and CD4(+) regulatory T cells led us to investigate the impact of IL-15 on the de novo generation and function of regulatory T cells in humans. Our data indicate that IL-15 does not counteract, but rather promotes the effect of TGF-beta on the de novo generation of regulatory T cells (Treg). Thus, in the presence of TGF-beta, IL-15 enhanced the acquisition of regulatory functions by CD4(+)CD25(-) T cells stimulated by anti-CD3 and anti-CD28 Abs. In contrast, IL-15 impaired the functions of Tregs by acting on effector CD4 and CD8 T cells. Accordingly, in the presence of IL-15, proliferation and IFN-gamma production by peripheral CD4 and CD8 T cells could not be efficiently inhibited by Tregs. IL-15-induced resistance of effector T cells to Tregs resulted from activation of the PI3K signaling pathway but did not involve the rescue of effector T cells from apoptosis. Altogether, these data point to the ambiguous role of IL-15 in the control of Treg functions. This dual role may be instrumental to mount rapid but transient proinflammatory immune responses against pathogens but may become deleterious in situations associated with protracted IL-15 over-expression. PMID:19454671

  14. Adhesion of ZAP-70+ chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway.

    PubMed

    Lafarge, Sandrine T; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2014-01-01

    CLL cell survival and proliferation is enhanced through direct contact with supporting cells present in lymphoid tissues. PI3Ks are critical signal transduction enzymes controlling B cell survival and activation. PI3K inhibitors have entered clinical trials and show promising therapeutic activity; however, it is unclear whether PI3K inhibitor drugs differentially affect ZAP-70 positive versus negative CLL cells or target specific microenvironmental interactions. Here we provide evidence that CD40L+IL-4, IL-8 or IL-6 enhance adhesion to stromal cells, with IL-6 showing a selective effect on ZAP-70 positive cells. Stimulatory effects of IL-8 or IL-6 are fully reversed by PI3K inhibition, while the effects of CD40L+IL-4 are partially reversed. While CD40L+IL-4 is the only stimulation increasing CLL cell survival for all patient groups, IL-6 protects ZAP-70 positive cells from cell death induced by PI3K inhibition. Altogether, our results indicate that targeting the PI3K pathway can reverse protective CLL-microenvironment interactions in both ZAP-70 positive and negative CLL despite their differences in cytokine responsiveness. PMID:23981382

  15. Inactivation of the phosphatidylinositol 3-kinase/Akt pathway is involved in BMP9-mediated tumor-suppressive effects in gastric cancer cells.

    PubMed

    Duan, Liang; Ye, Liwei; Wu, Rui; Wang, Haiyan; Li, Xueru; Li, Huan; Yuan, Shimei; Zha, He; Sun, Hui; Zhang, Yunyuan; Chen, Xian; Zhang, Yan; Zhou, Lan

    2015-06-01

    Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily signaling factors. Expression of several BMPs (BMP2, BMP4, and BMP7) is correlated to poor prognosis in gastric cancer patients. The function of BMP9, the latest discovered and most powerful osteogenetic factor, in gastric cancer is relatively unclear. In this report, we investigated the expression, function and underlying molecular mechanisms of BMP9 in gastric cancer. The results show that BMP9 expression was markedly decreased in gastric cancer tissues and cell lines. Enforced BMP9 expression in the gastric cancer cell lines SGC-7901 and MNK-45 increased apoptosis and reduced viability and migration. The in vivo function of BMP9 was evaluated in a xenograft mouse model. Tumors derived from SGC-7901 cells with enforced BMP9 expression (SGC-7901/BMP9) showed significantly reduced size and weight compared to that from control cells. Enforced BMP9 expression resulted in decreased Akt activity shown as lower levels of phosphorylation at Ser473 and Thr308 in Akt. The PI3K/Akt inhibitor LY294002 potentiated BMP9's viability and migration suppression, and apoptosis induction, which was associated with reduced expression of snail and VEGF and increased expression of E-cadherin. In addition, tumors derived from SGC-7901/BMP9 showed reduced Akt activity and VEGF expression, and increased E-cadherin expression. Therefore, our studies reveal for the first time that inhibition of the PI3K-Akt pathway is involved in the tumor suppressor effects of BMP9 in gastric cancer. PMID:25640278

  16. Protective effects of Phyllanthus emblica against myocardial ischemia-reperfusion injury: the role of PI3-kinase/glycogen synthase kinase 3β/β-catenin pathway.

    PubMed

    Thirunavukkarasu, Mahesh; Selvaraju, Vaithinathan; Tapias, Leonidas; Sanchez, Juan A; Palesty, J Alexander; Maulik, Nilanjana

    2015-12-01

    Clinical studies of Phyllanthus emblica (P. emblica) have shown that it increases production of nitric oxide, glutathione, and high-density lipoprotein (HDL); decreases low-density lipoprotein (LDL), total cholesterol, triglycerides, and high-sensitivity C-reactive protein (hsCRP); and significantly inhibits platelet aggregation. The following study was designed to examine the effect of P. emblica treatment on myocardial ischemia-reperfusion (I/R) injury and identify the molecular targets and its underlying mechanism(s). Experimental animals were divided into four groups: control sham (CS), P. emblica sham (PS), control I/R (CIR), and P. emblica I/R (PIR). Rats in the P. emblica groups were gavaged with aqueous P. emblica solution (100 mg/kg body weight) for 30 days. After 30 days of gavaging, the I/R group underwent I/R surgery (45-min ischemia) followed by 4 or 30 days of reperfusion. Rats in the sham group underwent surgery without ligation. Left ventricular tissue samples, 4 and 30 days after I/R, were used for Western blot analysis and immunohistochemistry, respectively. Western blot analysis showed upregulation of phosphorylated Akt and GSK3-β and increased nuclear translocation of β-catenin in the PIR group versus CIR. PIR rats also indicated reduced 3-nitrotyrosine and Caspase-3 expression. Increased phosphorylation of endothelial nitric oxide synthase (p-eNOS) and upregulation of anti-apoptotic protein Bcl-2 were found in the PIR group. Echocardiography showed increased ejection fraction and fractional shortening and decreased left ventricular internal diameter in experimental subjects compared to controls. There was decreased fibrosis in P. emblica-treated rats compared to controls. The results of this study indicate that P. emblica is capable of upregulating the PI3K/Akt/GSK3β/β-catenin cardioprotective pathway, thereby preserving cardiac tissue during ischemia-reperfusion injury. PMID:26342597

  17. Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity

    PubMed Central

    Gaelzer, Mariana Maier; Coelho, Bárbara Paranhos; de Quadros, Alice Hoffmann; Hoppe, Juliana Bender; Terra, Silvia Resende; Guerra, Maria Cristina Barea; Usach, Vanina; Guma, Fátima Costa Rodrigues; Gonçalves, Carlos Alberto Saraiva; Setton-Avruj, Patrícia; Battastini, Ana Maria Oliveira; Salbego, Christianne Gazzana

    2016-01-01

    Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent. PMID:27123999

  18. The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.

    PubMed

    Balakrishnan, K; Peluso, M; Fu, M; Rosin, N Y; Burger, J A; Wierda, W G; Keating, M J; Faia, K; O'Brien, S; Kutok, J L; Gandhi, V

    2015-09-01

    The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in chronic lymphocytic leukemia (CLL). The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR-stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; P<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15; P<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose-responsive inhibition of pAKT(Ser473) is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR-induced chemokines CCL3 and CCL4 secretion to 17% and 37%, respectively. Pre-treatment with 1 μM IPI-145 inhibits the chemotaxis toward CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR-activated signaling proteins AKT(Ser473), BAD(Ser112), ERK(Thr202/Tyr204) and S6(Ser235/236) are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B cells, sparing normal B- and T-lymphocytes. PMID:25917267

  19. [ROLE PHOSPHOINOSITID SIGNALING PATHWAY IN OPIOIDS CONTROL OF P2X3 RECEPTORS IN THE PRIMARY SENSORY NEURONS].

    PubMed

    Kulyk, V B; Chizhmakov, I V; Volkova, T M; Maximyuk, O P; Krishtal, O A

    2015-01-01

    Homomeric P2X3 receptors expressed in primary nociceptive neurons are crucial elements in the pain signal generation. In turn, opioid system regulates the intensity of this signal in both CNS and PNS. Here we describe the effects of opioids on P2X3 receptors in DRG neurons studied by using patch clamp technique. Activation of G-protein coupled opioid receptors by endogenous opioid Leu-enkephalin (Leu), resulted in the two opposite effects on P2X3 receptor-mediated currents (P2X3 currents). In particular, application of 1 µM Leu lead to the complete inhibition of P2X3 currents. However, after pretreatment of the neurons with a Gi/o-protein inhibitor pertussis toxin (PT), the same concentration of Leu caused facilitation of P2X3 currents. PLC inhibitor U-73122 at concentration of 1 µM completely eliminated both facilitating and inhibitory effects of Leu on P2X3 currents. Thus, opioid receptor agonists cause two oppositely directed effects on P2X3 receptors in DRG neurons of rats and both of them are mediated through PLC signaling pathway. Our results point to a possible molecular basis of the mechanism for the well-known transition inhibitory action of opioids (analgesia) to facilitating (hyperalgesia). PMID:26552301

  20. Channelopathies linked to plasma membrane phosphoinositides

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul

    2014-01-01

    The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

  1. Effects of lactic acid bacteria on cardiac apoptosis are mediated by activation of the phosphatidylinositol-3 kinase/AKT survival-signalling pathway in rats fed a high-fat diet.

    PubMed

    Wang, Hsueh-Fang; Lin, Pei-Pei; Chen, Chun-Hua; Yeh, Yu-Lan; Huang, Chun-Chih; Huang, Chih-Yang; Tsai, Cheng-Chih

    2015-02-01

    Through a high-fat diet, obesity leads to cardiomyocyte dysfunction and apoptosis. In addition, there is no evidence that probiotics have potential health effects associated with cardiac apoptosis in obese rats. The present study aimed to explore the effects of probiotics on obesity and cardiac apoptosis in rats fed a high-fat diet (HF). Eight‑week‑old male Wistar rats were separated randomly into five equally sized experimental groups: Normal diet (NC) and high-fat diet (HFC) groups, and high-fat diet supplemented with low (HFL), medium (HFM) or high (HFH) doses of multi‑strain probiotics groups. The rats were subsequently studied for 8 weeks. Food intake and body weights were recorded following sacrifice, and food utilization rates, body fat and serum cholesterol levels were analysed. The myocardial architecture of the left ventricle was evaluated by hematoxylin‑eosin staining, and key apoptotic‑related pathway molecules were analysed by western blotting. Rat weights and triglyceride levels were decreased with oral administration of high doses of probiotics (HFH) compared to the HFC group. Abnormal myocardial architecture and enlarged interstitial spaces were observed in HFC hearts, but were significantly decreased in groups that were provided multi‑strain probiotics compared with NC hearts. Western blot analysis demonstrated that key components of the Fas receptor‑ and mitochondrial‑dependent apoptotic pathways were significantly suppressed in multi‑strain probiotic treated groups compared to the HF group. Additionally, cardiac insulin, such as the insulin‑like growth factor I receptor (IGFIR)‑dependent survival signalling components, were highly induced in left ventricles from rats administered probiotics. Together, these findings strongly suggest that oral administration of probiotics may attenuate cardiomyocyte apoptosis by activation of the phosphatidylinositol‑3 kinase/AKT survival‑signalling pathway in obese rats. PMID:25484003

  2. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase

    PubMed Central

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-01-01

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K. PMID:15282551

  3. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

    2000-01-01

    Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

  4. Regulation of constitutive vascular endothelial growth factor secretion in retinal pigment epithelium/choroid organ cultures: p38, nuclear factor kappaB, and the vascular endothelial growth factor receptor-2/phosphatidylinositol 3 kinase pathway

    PubMed Central

    Westhues, Daniel; Lassen, Jens; Bartsch, Sofia; Roider, Johann

    2013-01-01

    Purpose The retinal pigment epithelium (RPE) is a major source of vascular endothelial growth factor (VEGF) in the eye. Despite the role of VEGF in ocular pathology, VEGF is an important factor in maintaining the choroid and the RPE. Accordingly, the VEGF is constitutively expressed in RPE. In this study, the regulation of constitutive VEGF expression was investigated in an RPE/choroid organ culture. Methods To investigate VEGF regulation, RPE/choroid of porcine origin were used. VEGF content was evaluated with enzyme-linked immunosorbent assay. The influence of several molecular factors was assessed with commercially available inhibitors (SU1498, bisindolylmaleimide, LY294002, nuclear factor kappaB [NFkB] activation inhibitor, mithramycin, YC-1, Stattic, SB203580). For toxicity measurements of inhibitors, primary RPE cells of porcine origin were used, and toxicity was evaluated with methyl thiazolyl tetrazolium assay. Results VEGF secretion as measured in the RPE/choroid organ culture was diminished after long-term (48 h) inhibition of vascular endothelial growth factor receptor-2 by VEGFR-2-antagonist SU1498. VEGF secretion was also diminished after phosphatidylinositol 3 kinase was inhibited by LY294002 for 48 h. Coapplication of the substances did not show an additive effect, suggesting that they use the same pathway in an autocrine-positive VEGF regulation loop. Inhibition of protein kinase C by bisindolylmaleimide, on the other hand, did not influence VEGF secretion in organ culture. Inhibition of the transcription factor SP-1 by mithramycin displayed effects after 24 h and 48 h. Inhibiting hypoxia-inducible factor-1 (HIF-1) and Stat3 did not show any influence on constitutive VEGF secretion. Inhibition of the transcription factor NFkB diminished VEGF secretion after 6 h (earliest measured time point) and remained diminished at all measured time points (24 h, 48 h). The same pattern was found when the inhibitor of mitogen-activated kinase p38 was applied. A

  5. Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts.

    PubMed

    Yen, Men Luh; Su, Jen Liang; Chien, Chung Liang; Tseng, Kuang Wen; Yang, Ching Yao; Chen, Wei Fang; Chang, Chiao Chia; Kuo, Min Liang

    2005-10-01

    Diosgenin, extracted from the root of wild yam (Dioscorea villosa), has been reported to demonstrate an opportunity for medical application. Vascular endothelial growth factor-A (VEGF-A) plays an important role in bone-related angiogenesis, a critical process occurring during bone formation and fracture healing. In this study, we examine whether diosgenin is able to induce VEGF-A expression and to promote angiogenesis in osteoblasts. For murine MC3T3-E1 preosteoblast-like cells, VEGF-A mRNA and protein expression seemed to be significantly elevated in response to diosgenin in a concentration-dependent fashion. Conditioned media prepared from cells treated with diosgenin induced strong angiogenic activity in either in vitro or ex vivo angiogenesis assay. Furthermore, diosgenin treatment increased the stability and activity of HIF-1alpha protein. Inhibition of HIF-1alpha activity by transfection with DN-HIF-1alpha significantly diminished diosgenin-mediated VEGF-A up-regulation. The use of pharmacological inhibitors or genetic inhibition revealed that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways were potentially required for diosgenin-induced HIF-1 activation and subsequent VEGF-A up-regulation. It is noteworthy that an estrogen receptor binding assay revealed that diosgenin has the strong ability to replace [(3)H]estradiol bound to estrogen receptor (IC(50), 10 nM). In addition, the specific estrogen receptor antagonists ICI 182,780 (faslodex) and tamoxifen were noted to be able to strongly inhibit diosgenin-induced, src kinase-dependent Akt and p38 MAPK activation. Taken together, such results provide evidence that diosgenin up-regulates VEGF-A and promotes angiogenesis in preosteoblast-like cells by a hypoxia-inducible factor-1alpha-dependent mechanism involving the activation of src kinase, p38 MAPK, and Akt signaling pathways via estrogen receptor. PMID:15998873

  6. o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

    SciTech Connect

    Han, Eun Hee; Kim, Ji Young; Kim, Hyung-Kyun; Hwang, Yong Pil; Jeong, Hye Gwang

    2008-12-01

    Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p'-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p'-DDT markedly enhanced the production of prostaglandin E{sub 2} (PGE{sub 2}), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p'-DDT dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p'-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-{kappa}B site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p'-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p'-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p'-DDT.

  7. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

    PubMed Central

    Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

    2015-01-01

    Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

  8. D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor.

    PubMed Central

    Whiteford, C C; Best, C; Kazlauskas, A; Ulug, E T

    1996-01-01

    Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) alpha-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agoniststimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells. PMID:8920990

  9. Synthesis and Function of Membrane Phosphoinositides in Budding Yeast, Saccharomyces cerevisiae

    PubMed Central

    Strahl, Thomas; Thorner, Jeremy

    2007-01-01

    It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast. PMID:17382260

  10. Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases.

    PubMed

    Nagahara, Yukitoshi; Nagamori, Taome; Tamegai, Hidekazu; Hitokuwada, Mami; Yoshimi, Yoji; Ikekita, Masahiko; Shinomiya, Takahisa

    2011-01-01

    Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation. PMID:22038771

  11. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response

    PubMed Central

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-01-01

    Abstract Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

  12. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

    PubMed

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-09-01

    Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

  13. Phosphoinositides in the mammalian endo-lysosomal network

    PubMed Central

    Cullen, Peter J.; Carlton, Jeremy G.

    2014-01-01

    The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform. PMID:22374088

  14. Autoradiographic imaging of phosphoinositide turnover in the brain

    SciTech Connect

    Hwang, P.M.; Bredt, D.S.; Snyder, S.H. )

    1990-08-17

    With ({sup 3}H)cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product ({sup 3}H)cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

  15. Cholesterol stabilizes fluid phosphoinositide domains

    PubMed Central

    Jiang, Zhiping; Redfern, Roberta E.; Isler, Yasmin; Ross, Alonzo H.

    2014-01-01

    Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al. Biochemistry 48, (2009) 9360). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of

  16. Activation of phosphatidylinositol 3-kinase by insulin.

    PubMed Central

    Ruderman, N B; Kapeller, R; White, M F; Cantley, L C

    1990-01-01

    Insulin action appears to require the protein-tyrosine kinase domain of the beta subunit of the insulin receptor. Despite this, the identities and biochemical functions of the cellular targets of this tyrosine kinase are unknown. A phosphatidylinositol 3-kinase (PI 3-kinase) that phosphorylates the D-3 position of the inositol ring associates with several protein-tyrosine kinases. Here we report that PI 3-kinase activity is immunoprecipitated from insulin-stimulated CHO cells by antiphosphotyrosine and anti-insulin receptor antibodies. Insulin as low as 0.3 nM increased immunoprecipitable PI 3-kinase activity within 1 min. Increases in activity were much greater in CHO cells expressing the human insulin receptor (100,000 receptors per cell) than in control CHO cells (2000 receptors per cell). During insulin stimulation, various lipid products of the PI 3-kinase either appeared or increased in quantity in intact cells, suggesting that the appearance of immunoprecipitable PI 3-kinase reflects an increase in its activity in vivo. These results indicate that insulin at physiological concentrations regulates the PI 3-kinase and suggest that this regulation involves a physical association between the insulin receptor and the PI 3-kinase and tyrosyl phosphorylation. Images PMID:2154747

  17. Ligand-based Pharmacophore Modeling; Atom-based 3D-QSAR Analysis and Molecular Docking Studies of Phosphoinositide-Dependent Kinase-1 Inhibitors

    PubMed Central

    Kirubakaran, P.; Muthusamy, K.; Singh, K. H. D.; Nagamani, S.

    2012-01-01

    Phosphoinositide-dependent kinase-1 plays a vital role in the PI3-kinase signaling pathway that regulates gene expression, cell cycle growth and proliferation. The common human cancers include lung, breast, blood and prostate possess over stimulation of the phosphoinositide-dependent kinase-1 signaling and making phosphoinositide-dependent kinase-1 an interesting therapeutic target in oncology. A ligand-based pharmacophore and atom-based 3D-QSAR studies were carried out on a set of 82 inhibitors of PDK1. A six point pharmacophore with two hydrogen bond acceptors (A), three hydrogen bond donors (D) and one hydrophobic group (H) was obtained. The pharmacophore hypothesis yielded a 3D-QSAR model with good partial least square statistics results. The training set correlation is characterized by partial least square factors (R2 = 0.9557, SD = 0.2334, F = 215.5, P = 1.407e-32). The test set correlation is characterized by partial least square factors (Q2 ext = 0.7510, RMSE = 0.5225, Pearson-R =0.8676). The external validation indicated that our QSAR model possess high predictive power with good value of 0.99 and value of 0.88. The docking results show the binding orientations of these inhibitors at active site amino acid residues (Ala162, Thr222, Glu209 and Glu166) of phosphoinositide-dependent kinase-1 protein. The binding free energy interactions of protein-ligand complex have been calculated, which plays an important role in molecular recognition and drug design approach. PMID:23325995

  18. Phosphoinositide Phosphatases in Cell Biology and Disease

    PubMed Central

    Liu, Yang; Bankaitis, Vytas A.

    2010-01-01

    Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

  19. Follicle-stimulating Hormone Activation of Hypoxia-inducible Factor-1 by the Phosphatidylinositol 3-Kinase/AKT/Ras Homolog Enriched in Brain (Rheb)/Mammalian Target of Rapamycin (mTOR) Pathway Is Necessary for Induction of Select Protein Markers of Follicular Differentiation*

    PubMed Central

    Alam, Hena; Maizels, Evelyn T.; Park, Youngkyu; Ghaey, Shail; Feiger, Zachary J.; Chandel, Navdeep S.; Hunzicker-Dunn, Mary

    2006-01-01

    We sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-α, microtubule-associated protein 2D, and the PKA type IIβ regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farne-syltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-α, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1α interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers. PMID:14982927

  20. Structural, Biochemical, and Biophysical Characterization of Idelalisib Binding to Phosphoinositide 3-Kinase δ*

    PubMed Central

    Somoza, John R.; Koditek, David; Villaseñor, Armando G.; Novikov, Nikolai; Wong, Melanie H.; Liclican, Albert; Xing, Weimei; Lagpacan, Leanna; Wang, Ruth; Schultz, Brian E.; Papalia, Giuseppe A.; Samuel, Dharmaraj; Lad, Latesh; McGrath, Mary E.

    2015-01-01

    Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib. PMID:25631052

  1. Frequency and amplitude control of cortical oscillations by phosphoinositide waves.

    PubMed

    Xiong, Ding; Xiao, Shengping; Guo, Su; Lin, Qingsong; Nakatsu, Fubito; Wu, Min

    2016-03-01

    Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction. PMID:26751515

  2. AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity.

    PubMed

    Yoo, Cheol-Min; Quan, Li; Cannon, Ashley E; Wen, Jiangqi; Blancaflor, Elison B

    2012-03-01

    The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor-gtpase-activating protein (ARF-GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked-out in a gene encoding a phosphatidylinositol-4-phosphate (PI-4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol-4-phosphate 5-kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair-expressed vegetative actin isoform. Consistent with our double-mutant studies, targeting of tip growth components involved in PI signaling (PI-4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺](cyt) ) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal-, [Ca²⁺](cyt-) , ROP2-, and RABA4b-mediated root hair development. PMID:22098134

  3. The role of class I, II and III PI 3-kinases in platelet production and activation and their implication in thrombosis.

    PubMed

    Valet, Colin; Severin, Sonia; Chicanne, Gaëtan; Laurent, Pierre Alexandre; Gaits-Iacovoni, Frédérique; Gratacap, Marie-Pierre; Payrastre, Bernard

    2016-05-01

    Blood platelets play a pivotal role in haemostasis and are strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their critical role in pathophysiology, platelets represent a valuable model to investigate, both in vitro and in vivo, the biological roles of different branches of the phosphoinositide metabolism, which is highly active in platelets. While the phospholipase C (PLC) pathway has a crucial role in platelet activation, it is now well established that at least one class I phosphoinositide 3-kinase (PI3K) is also mandatory for proper platelet functions. Except class II PI3Kγ, all other isoforms of PI3Ks (class I α, β, γ, δ; class II α, β and class III) are expressed in platelets. Class I PI3Ks have been extensively studied in different models over the past few decades and several isoforms are promising drug targets to treat cancer and immune diseases. In platelet activation, it has been shown that while class I PI3Kδ plays a minor role, class I PI3Kβ has an important function particularly in thrombus growth and stability under high shear stress conditions found in stenotic arteries. This class I PI3K is a potentially interesting target for antithrombotic strategies. The role of class I PI3Kα remains ill defined in platelets. Herein, we will discuss our recent data showing the potential impact of inhibitors of this kinase on thrombus formation. The role of class II PI3Kα and β as well as class III PI3K (Vps34) in platelet production and function is just emerging. Based on our data and those very recently published in the literature, we will discuss the impact of these three PI3K isoforms in platelet production and functions and in thrombosis. PMID:26714793

  4. Infectious bursal disease virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by interaction of VP5 protein with the p85{alpha} subunit of PI3K

    SciTech Connect

    Wei Li; Hou Lei; Zhu Shanshan; Wang Jing; Zhou Jiao; Liu Jue

    2011-08-15

    Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is commonly activated upon virus infection and has been implicated in the regulation of diverse cellular functions such as proliferation and apoptosis. The present study demonstrated for the first time that infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, can induce Akt phosphorylation in cultured cells, by a mechanism that is dependent on PI3K. Inhibition of PI3K activation greatly enhanced virus-induced cytopathic effect and apoptotic cell death as evidenced by cleavage of poly-ADP ribose polymerase and activation of caspase-3. Investigations into the mechanism of PI3K/Akt activation revealed that IBDV activates PI3K/Akt signaling through binding of the non-structural protein VP5 to regulatory subunit p85{alpha} of PI3K resulting in the suppression of premature apoptosis and improved virus growth after infection. The results presented here provide a basis for understanding molecular mechanism of IBDV infection.

  5. Rational targeting of BRAF and PI3-Kinase signaling for melanoma therapy

    PubMed Central

    Deuker, Marian M.; McMahon, Martin

    2016-01-01

    ABSTRACT Although mitogen-activated protein kinase (MAPK) inhibitors elicit initial regression of BRAF-mutated melanoma, drug resistance is an inevitable and fatal event. We recently reported that in genetically engineered mouse models of BRAF-mutated melanoma, isoform-selective phosphatidylinositol 3-kinase inhibition cooperates with MAPK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance. PMID:27314067

  6. Rational targeting of BRAF and PI3-Kinase signaling for melanoma therapy.

    PubMed

    Deuker, Marian M; McMahon, Martin

    2016-05-01

    Although mitogen-activated protein kinase (MAPK) inhibitors elicit initial regression of BRAF-mutated melanoma, drug resistance is an inevitable and fatal event. We recently reported that in genetically engineered mouse models of BRAF-mutated melanoma, isoform-selective phosphatidylinositol 3-kinase inhibition cooperates with MAPK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance. PMID:27314067

  7. Phosphoinositide signaling in somatosensory neurons.

    PubMed

    Rohacs, Tibor

    2016-05-01

    Somatosensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) are responsible for detecting thermal and tactile stimuli. They are also the primary neurons mediating pain and itch. A large number of cell surface receptors in these neurons couple to phospholipase C (PLC) enzymes leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the generation of downstream signaling molecules. These neurons also express many different ion channels, several of which are regulated by phosphoinositides. This review will summarize the knowledge on phosphoinositide signaling in DRG neurons, with special focus on effects on sensory and other ion channels. PMID:26724974

  8. Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-kappaB-dependent and -independent pathways.

    PubMed

    Zheng, Xueyan; Karsan, Aly; Duronio, Vincent; Chu, Fanny; Walker, David C; Bai, Tony R; Schellenberg, R Robert

    2002-11-01

    Basophils are key effector cells of allergic reactions. Although proinflammatory cytokines, such as interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5, inhibit eosinophil apoptosis in vitro, little is known about basophil apoptosis, and the signalling mechanisms required for basophil survival remain undefined. To address this issue, we used a novel negative-selection system to isolate human basophils to a purity of > 95%, and evaluated apoptosis by morphology using light and transmission electron microscopy, and by annexin-V binding and propidium iodide incorporation using flow cytometry. In this study, we demonstrated that the spontaneous rate of apoptotic basophils was higher than that of eosinophils as, at 24 hr, 57.6 +/- 4.7% of basophils underwent apoptosis compared with 39.5 +/- 3.8% of eosinophils. In addition, basophil cell death was significantly inhibited when cultured with IL-3 for 48 hr (84.6 +/- 4.9% vehicle-treated cells versus 40.9 +/- 3.9% IL-3-treated cells). IL-3 also up-regulated basophil CD69 surface expression. The effects of IL-3 on apoptosis and CD69 surface expression of human basophils were completely blocked by LY294002 (LY), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K), but only partially inhibited by lactacystin, a proteasome inhibitor that prevents degradation of IkappaB and NF-kappaB translocation. These observations reveal the novel finding that IL-3 prevents basophil apoptosis through the activation of PI3-K, which is only partially NF-kappaB dependent. As basophils are active participants in allergic reactions and IL-3 is one of the abundant proinflammatory cytokines in secretions from allergic tissue, we suggest that IL-3-mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders. PMID:12423306

  9. Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis

    PubMed Central

    Aggarwal, Chhavi; Łabuz, Justyna; Gabryś, Halina

    2013-01-01

    Phototropins are UVA/blue-light receptors involved in controlling the light-dependent physiological responses which serve to optimize the photosynthetic activity of plants and promote growth. The phototropin-induced phosphoinositide (PI) metabolism has been shown to be essential for stomatal opening and phototropism. However, the role of PIs in phototropin-induced chloroplast movements remains poorly understood. The aim of this work is to determine which PI species are involved in the control of chloroplast movements in Arabidopsis and the nature of their involvement. We present the effects of the inactivation of phospholipase C (PLC), PI3-kinase (PI3K) and PI4-kinase (PI4K) on chloroplast relocations in Arabidopsis. The inhibition of the phosphatidylinositol 4,5-bisphospahte [PI(4,5)P2]-PLC pathway, using neomycin and U73122, suppressed the phot2-mediated chloroplast accumulation and avoidance responses, without affecting movement responses controlled by phot1. On the other hand, PI3K and PI4K activities are more restricted to phot1- and phot2-induced weak-light responses. The inactivation of PI3K and PI4K by wortmannin and LY294002 severely affected the weak blue-light-activated accumulation response but had little effect on the strong blue-light-activated avoidance response. The inhibitory effect observed with PI metabolism inhibitors is, at least partly, due to a disturbance in Ca2+(c) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca2+(c) rise. These results demonstrate the importance of PIs in chloroplast movements, with the PI(4,5)P2-PLC pathway involved in phot2 signaling while PI3K and PI4K are required for the phot1- and phot2-induced accumulation response. Our results suggest that these PIs modulate cytosolic Ca2+ signaling during movements. PMID:23405144

  10. VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS

    PubMed Central

    Balla, Tamas; Várnai, Péter

    2011-01-01

    This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

  11. Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase.

    PubMed Central

    Chellaiah, M; Hruska, K

    1996-01-01

    Based on previous studies demonstrating activation of phosphatidylinositol 3-hydroxyl kinase (PI3-kinase) and stimulation of a change in cell shape, we examined the effect of osteopontin on the association of phospholipids with gelsolin, an actin-capping/severing protein. Osteopontin stimulated a rapid increase in phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate levels associated with gelsolin in Triton-soluble fractions of cell lysates. The increased levels of phosphatidylinositol triphosphate associated with gelsolin were due to stimulation of PI3-kinase activity associated with gelsolin in the Triton-soluble fractions, and they were blocked by the PI3-kinase inhibitor wortmannin. Osteopontin stimulated translocation of PI3-kinase from the Triton-insoluble to Triton-soluble gelsolin. Osteopontin also decreased Triton-soluble gelsolin/actin complexes consistent with actin uncapping, and increased F-actin levels, which were also blocked by wortmannin. The osteopontin effects were mediated through binding to the alpha(v)beta 3 integrin. Taken together, our studies indicate that osteopontin/alpha(v)beta 3-mediated changes in gelsolin-associated phosphoinositide levels and PI3-kinase activity are related to stimulation of F-actin formation in osteoclasts. Images PMID:8744948

  12. Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway.

    PubMed

    Tiwari, Shashi Kant; Seth, Brashket; Agarwal, Swati; Yadav, Anuradha; Karmakar, Madhumita; Gupta, Shailendra Kumar; Choubey, Vinay; Sharma, Abhay; Chaturvedi, Rajnish Kumar

    2015-11-20

    Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling. PMID:26420483

  13. Frequent phosphatidylinositol-3-kinase mutations in proliferative breast lesions.

    PubMed

    Ang, Daphne C; Warrick, Andrea L; Shilling, Amy; Beadling, Carol; Corless, Christopher L; Troxell, Megan L

    2014-05-01

    The phosphatidylinositol-3-kinase pathway is one of the most commonly altered molecular pathways in invasive breast carcinoma, with phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) mutations in 25% of invasive carcinomas. Ductal carcinoma in situ (DCIS), benign papillomas, and small numbers of columnar cell lesions harbor an analogous spectrum of PIK3CA and AKT1 mutations, yet there is little data on usual ductal hyperplasia and atypical ductal and lobular neoplasias. We screened 192 formalin-fixed paraffin-embedded breast lesions from 75 patients for point mutations using a multiplexed panel encompassing 643 point mutations across 53 genes, including 58 PIK3CA substitutions. PIK3CA point mutations were identified in 31/62 (50%) proliferative lesions (usual ductal hyperplasia and columnar cell change), 10/14 (71%) atypical hyperplasias (atypical ductal hyperplasia and flat epithelial atypia), 7/16 (44%) lobular neoplasias (atypical lobular hyperplasia and lobular carcinoma in situ), 10/21 (48%) DCIS, and 13/37 (35%) invasive carcinomas. In genotyping multiple lesions of different stage from the same patient/specimen, we found considerable heterogeneity; most notably, in 12 specimens the proliferative lesion was PIK3CA mutant but the concurrent carcinoma was wild type. In 11 additional specimens, proliferative epithelium and cancer contained different point mutations. The frequently discordant genotypes of usual ductal hyperplasia/columnar cell change and concurrent carcinoma support a role for PIK3CA-activating point mutations in breast epithelial proliferation, perhaps more so than transformation. Further, these data suggest that proliferative breast lesions are heterogeneous and may represent non-obligate precursors of invasive carcinoma. PMID:24186142

  14. Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

    Cancer.gov

    In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

  15. HGF/c-Met signaling promotes liver progenitor cell migration and invasion by an epithelial-mesenchymal transition-independent, phosphatidyl inositol-3 kinase-dependent pathway in an in vitro model.

    PubMed

    Suárez-Causado, A; Caballero-Díaz, D; Bertrán, E; Roncero, C; Addante, A; García-Álvaro, M; Fernández, M; Herrera, B; Porras, A; Fabregat, I; Sánchez, A

    2015-10-01

    Oval cells constitute an interesting hepatic cell population. They contribute to sustain liver regeneration during chronic liver damage, but in doing this they can be target of malignant conversion and become tumor-initiating cells and drive hepatocarcinogenesis. The molecular mechanisms beneath either their pro-regenerative or pro-tumorigenic potential are still poorly understood. In this study, we have investigated the role of the HGF/c-Met pathway in regulation of oval cell migratory and invasive properties. Our results show that HGF induces c-Met-dependent oval cell migration both in normal culture conditions and after in vitro wounding. HGF-triggered migration involves F-actin cytoskeleton reorganization, which is also evidenced by activation of Rac1. Furthermore, HGF causes ZO-1 translocation from cell-cell contact sites to cytoplasm and its concomitant activation by phosphorylation. However, no loss of expression of cell-cell adhesion proteins, including E-cadherin, ZO-1 and Occludin-1, is observed. Additionally, migration does not lead to cell dispersal but to a characteristic organized pattern in rows, in turn associated with Golgi compaction, providing strong evidence of a morphogenic collective migration. Besides migration, HGF increases oval cell invasion through extracellular matrix, a process that requires PI3K activation and is at least partly mediated by expression and activation of metalloproteases. Altogether, our findings provide novel insights into the cellular and molecular mechanisms mediating the essential role of HGF/c-Met signaling during oval cell-mediated mouse liver regeneration. PMID:26001768

  16. An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

    PubMed Central

    Jiang, Bing-Hua; Zheng, Jenny Z.; Vogt, Peter K.

    1998-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis. PMID:9826674

  17. Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions

    SciTech Connect

    S Jackson; S Al-Saigh; C Schultz; M Junop

    2011-12-31

    PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.

  18. PI3K pathway alterations in cancer: variations on a theme

    PubMed Central

    Yuan, TL; Cantley, LC

    2012-01-01

    The high frequency of phosphoinositide 3-kinase (PI3K) pathway alterations in cancer has led to a surge in the development of PI3K inhibitors. Many of these targeted therapies are currently in clinical trials and show great promise for the treatment of PI3K-addicted tumors. These recent developments call for a re-evaluation of the oncogenic mechanisms behind PI3K pathway alterations. This pathway is unique in that every major node is frequently mutated or amplified in a wide variety of solid tumors. Receptor tyrosine kinases upstream of PI3K, the p110α catalytic subunit of PI3K, the downstream kinase, AKT, and the negative regulator, PTEN, are all frequently altered in cancer. In this review, we will examine the oncogenic properties of these genetic alterations to understand whether they are redundant or distinct and propose treatment strategies tailored for these genetic lesions. PMID:18794884

  19. Oncogenic activation of the PI3-kinase p110β isoform via the tumor-derived PIK3Cβ(D1067V) kinase domain mutation.

    PubMed

    Pazarentzos, E; Giannikopoulos, P; Hrustanovic, G; St John, J; Olivas, V R; Gubens, M A; Balassanian, R; Weissman, J; Polkinghorn, W; Bivona, T G

    2016-03-01

    Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs widely in human cancers. Although somatic mutations in the PI3K pathway genes PIK3CA and PTEN are known to drive PI3K pathway activation and cancer growth, the significance of somatic mutations in other PI3K pathway genes is less clear. Here, we establish the signaling and oncogenic properties of a recurrent somatic mutation in the PI3K p110β isoform that resides within its kinase domain (PIK3Cβ(D1067V)). We initially observed PIK3Cβ(D1067V) by exome sequencing analysis of an EGFR-mutant non-small cell lung cancer (NSCLC) tumor biopsy from a patient with acquired erlotinib resistance. On the basis of this finding, we hypothesized that PIK3Cβ(D1067V) might function as a novel tumor-promoting genetic alteration, and potentially an oncogene, in certain cancers. Consistent with this hypothesis, analysis of additional tumor exome data sets revealed the presence of PIK3Cβ(D1067V) at low frequency in other patient tumor samples (including renal cell carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, melanoma, thyroid carcinoma and endometrial carcinoma). Functional studies revealed that PIK3Cβ(D1067V) promoted PI3K pathway signaling, enhanced cell growth in vitro, and was sufficient for tumor formation in vivo. Pharmacologic inhibition of PIK3Cβ with TGX-221 (isoform-selective p110β inhibitor) specifically suppressed growth in patient-derived renal-cell carcinoma cells with endogenous PIK3Cβ(D1067V) and in NIH-3T3 and human EGFR-mutant lung adenocarcinoma cells engineered to express this mutant PI3K. In the EGFR-mutant lung adenocarcinoma cells, expression of PIK3Cβ(D1067V) also promoted erlotinib resistance. Our data establish a novel oncogenic form of PI3K, revealing the signaling and oncogenic properties of PIK3Cβ(D1067V) and its potential therapeutic relevance in cancer. Our findings provide new insight into the genetic mechanisms underlying PI3K pathway activation in

  20. EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3-kinase.

    PubMed Central

    Akimoto, K; Takahashi, R; Moriya, S; Nishioka, N; Takayanagi, J; Kimura, K; Fukui, Y; Osada, S i; Mizuno, K; Hirai, S i; Kazlauskas, A; Ohno, S

    1996-01-01

    Overexpression of a TPA-insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal-dependent changes in aKPClambda were observed for a PDGF receptor add-back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3-kinase, and were inhibited by wortmannin, an inhibitor of PI3-kinase. Furthermore, an N-terminal fragment of the catalytic subunit of PI3-kinase, p110alpha, inhibited aPKClambda-dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3-kinase. Images PMID:8631300

  1. A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis*S⃞

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Girard-Dias, Wendell; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Docampo, Roberto; Alonso, Guillermo D.

    2008-01-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking. PMID:18801733

  2. Phosphatidylinositol 3 kinase modulation of trophoblast cell differentiation

    PubMed Central

    2010-01-01

    Background The trophoblast lineage arises as the first differentiation event during embryogenesis. Trophoblast giant cells are one of several end-stage products of trophoblast cell differentiation in rodents. These cells are located at the maternal-fetal interface and are capable of invasive and endocrine functions, which are necessary for successful pregnancy. Rcho-1 trophoblast stem cells can be effectively used as a model for investigating trophoblast cell differentiation. In this report, we evaluated the role of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in the regulation of trophoblast cell differentiation. Transcript profiles from trophoblast stem cells, differentiated trophoblast cells, and differentiated trophoblast cells following disruption of PI3K signaling were generated and characterized. Results Prominent changes in gene expression accompanied the differentiation of trophoblast stem cells. PI3K modulated the expression of a subset of trophoblast cell differentiation-dependent genes. Among the PI3K-responsive genes were those encoding proteins contributing to the invasive and endocrine phenotypes of trophoblast giant cells. Conclusions Genes have been identified with differential expression patterns associated with trophoblast stem cells and trophoblast cell differentiation; a subset of these genes are regulated by PI3K signaling, including those impacting the differentiated trophoblast giant cell phenotype. PMID:20840781

  3. 3-Bromopyruvate induces apoptosis in breast cancer cells by downregulating Mcl-1 through the PI3K/Akt signaling pathway.

    PubMed

    Liu, Zhe; Zhang, Yuan-Yuan; Zhang, Qian-Wen; Zhao, Su-Rong; Wu, Cheng-Zhu; Cheng, Xiu; Jiang, Chen-Chen; Jiang, Zhi-Wen; Liu, Hao

    2014-04-01

    The hexokinase inhibitor 3-bromopyruvate (3-BrPA) can inhibit glycolysis in tumor cells to reduce ATP production, resulting in apoptosis. However, as 3-BrPA is an alkylating agent, its cytotoxic action may be induced by other molecular mechanisms. The results presented here reveal that 3-BrPA-induced apoptosis is caspase independent. Further, 3-BrPA induces the generation of reactive oxygen species in MDA-MB-231 cells, leading to mitochondria-mediated apoptosis. These results suggest that caspase-independent apoptosis may be induced by the generation of reactive oxygen species. In this study, we also demonstrated that 3-BrPA induces apoptosis through the downregulation of myeloid cell leukemia-1 (Mcl-1) in MDA-MB-231 breast cancer cells. The results of Mcl-1 knockdown indicate that Mcl-1 plays an important role in 3-BrPA-induced apoptosis. Further, the upregulation of Mcl-1 expression in 3-BrPA-treated MDA-MB-231 cells significantly increases cell viability. In addition, 3-BrPA treatment resulted in the downregulation of p-Akt, suggesting that 3-BrPA may downregulate Mcl-1 through the phosphoinositide-3-kinase/Akt pathway. These findings indicate that 3-BrPA induces apoptosis in breast cancer cells by downregulating Mcl-1 through the phosphoinositide-3-kinase/Akt signaling pathway. PMID:24492287

  4. Phosphoinositide 3-kinase γ plays a critical role in bleomycin-induced pulmonary inflammation and fibrosis in mice.

    PubMed

    Russo, Remo C; Garcia, Cristiana C; Barcelos, Lucíola S; Rachid, Milene A; Guabiraba, Rodrigo; Roffê, Ester; Souza, Adriano L S; Sousa, Lirlândia P; Mirolo, Massimiliano; Doni, Andrea; Cassali, Geovanni D; Pinho, Vanessa; Locati, Massimo; Teixeira, Mauro M

    2011-02-01

    PI3Kγ is central in signaling diverse arrays of cellular functions and inflammation. Pulmonary fibrosis is associated with pulmonary inflammation, angiogenesis, and deposition of collagen and is modeled by instillation of bleomycin. The role of PI3Kγ in mediating bleomycin-induced pulmonary inflammation and fibrosis in mice and potential mechanisms involved was investigated here. WT or PI3Kγ KO mice were instilled with bleomycin and leukocyte subtype influx, cytokine and chemokine levels, and angiogenesis and tissue fibrosis evaluated. The activation of lung-derived leukocytes and fibroblasts was evaluated in vitro. The relevance of PI3Kγ for endothelial cell function was evaluated in HUVECs. PI3Kγ KO mice had greater survival and weight recovery and less fibrosis than WT mice after bleomycin instillation. This was associated with decreased production of TGF-β(1) and CCL2 and increased production of IFN-γ and IL-10. There was reduced expression of collagen, fibronectin, α-SMA, and von Willebrand factor and decreased numbers and activation of leukocytes and phosphorylation of AKT and IκB-α. PI3Kγ KO mice had a reduced number and area of blood vessels in the lungs. In vitro, treatment of human endothelial cells with the PI3Kγ inhibitor AS605240 decreased proliferation, migration, and formation of capillary-like structures. AS605240 also decreased production of collagen by murine lung-derived fibroblasts. PI3Kγ deficiency confers protection against bleomycin-induced pulmonary injury, angiogenesis, and fibrosis through the modulation of leukocyte, fibroblast, and endothelial cell functions. Inhibitors of PI3Kγ may be beneficial for the treatment of pulmonary fibrosis. PMID:21048214

  5. Phosphoinositide 3-Kinase δ Regulates Dectin-2 Signaling and the Generation of Th2 and Th17 Immunity.

    PubMed

    Lee, Min Jung; Yoshimoto, Eri; Saijo, Shinobu; Iwakura, Yoichiro; Lin, Xin; Katz, Howard R; Kanaoka, Yoshihide; Barrett, Nora A

    2016-07-01

    The C-type lectin receptor Dectin-2 can trigger the leukotriene C4 synthase-dependent generation of cysteinyl leukotrienes and the caspase-associated recruitment domain 9- and NF-κB-dependent generation of cytokines, such as IL-23, IL-6, and TNF-α, to promote Th2 and Th17 immunity, respectively. Dectin-2 activation also elicits the type 2 cytokine IL-33, but the mechanism by which Dectin-2 induces these diverse innate mediators is poorly understood. In this study, we identify a common upstream requirement for PI3Kδ activity for the generation of each Dectin-2-dependent mediator elicited by the house dust mite species, Dermatophagoides farinae, using both pharmacologic inhibition and small interfering RNA knockdown of PI3Kδ in bone marrow-derived dendritic cells. PI3Kδ activity depends on spleen tyrosine kinase (Syk) and regulates the activity of protein kinase Cδ, indicating that PI3Kδ is a proximal Syk-dependent signaling intermediate. Inhibition of PI3Kδ also reduces cysteinyl leukotrienes and cytokines elicited by Dectin-2 cross-linking, confirming the importance of this molecule in Dectin-2 signaling. Using an adoptive transfer model, we demonstrate that inhibition of PI3Kδ profoundly reduces the capacity of bone marrow-derived dendritic cells to sensitize recipient mice for Th2 and Th17 pulmonary inflammation in response to D. farinae Furthermore, administration of a PI3Kδ inhibitor during the sensitization of wild-type mice prevents the generation of D. farinae-induced pulmonary inflammation. These results demonstrate that PI3Kδ regulates Dectin-2 signaling and its dendritic cell function. PMID:27194783

  6. B-cell receptor pathway modulators in NHL

    PubMed Central

    Blum, Kristie A.

    2016-01-01

    With the recent success of the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, and the phosphoinositide-3-kinase (PI3K) inhibitor, idelalisib, in the treatment of patients with relapsed or refractory non-Hodgkin's lymphoma (NHL), a number of new agents targeting the B-cell receptor (BCR) pathway are in clinical development. In addition, multiple trials combining these agents with conventional cytotoxic chemotherapy, immunomodulatory agents, monoclonal antibodies, or other kinase inhibitors are underway. This review will summarize the current data with the use of single agent and combination therapy with BCR inhibitors in NHL. In addition, commonly encountered as well as serious toxicities and hypothesized resistance mechanisms will be discussed. Lastly, this review will examine the future of these agents and opportunities to maneuver them into the front-line setting in selected NHL subtypes. PMID:26637705

  7. Spatial Sensing in Fibroblasts Mediated by 3′ Phosphoinositides

    PubMed Central

    Haugh, Jason M.; Codazzi, Franca; Teruel, Mary; Meyer, Tobias

    2000-01-01

    The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3′ PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein–tagged Akt pleckstrin homology domain (GFP–AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3′ PI lipids in the adhesion zone of fibroblasts. Polar GFP–AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3′ PI production and rapid membrane spreading implicates 3′ PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3′ PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 μm2/s) and short lifetime of 3′ PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3′ PI second messengers. PMID:11121441

  8. A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport.

    PubMed

    Gual, Philippe; Shigematsu, Satoshi; Kanzaki, Makoto; Grémeaux, Thierry; Gonzalez, Teresa; Pessin, Jeffrey E; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2002-11-15

    Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein). We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgamma1, the p85 subunit of the phosphoinositide 3-kinase and Crk-II. It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins. We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake. Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process. Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport. In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains. These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake. PMID:12215429

  9. The role of phosphoinositide-regulated actin reorganization in chemotaxis and cell migration

    PubMed Central

    Wu, C-Y; Lin, M-W; Wu, D-C; Huang, Y-B; Huang, H-T; Chen, C-L

    2014-01-01

    Reorganization of the actin cytoskeleton is essential for cell motility and chemotaxis. Actin-binding proteins (ABPs) and membrane lipids, especially phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 are involved in the regulation of this reorganization. At least 15 ABPs have been reported to interact with, or regulated by phosphoinositides (PIPs) whose synthesis is regulated by extracellular signals. Recent studies have uncovered several parallel intracellular signalling pathways that crosstalk in chemotaxing cells. Here, we review the roles of ABPs and phosphoinositides in chemotaxis and cell migration. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:25420930

  10. Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

    PubMed Central

    Maddika, S; Wiechec, E; Ande, SR; Poon, IK; Fischer, U; Wesselborg, S; Jans, DA; Schulze-Osthoff, K; Los, M

    2010-01-01

    Apoptin, a small protein from the chicken anemia virus, has attracted attention because of its specificity in killing tumor cells. Localization of apoptin in the nucleus of tumor cells has been shown to be vital for proapoptotic activity, however, targeted expression of apoptin in the nucleus of normal cells does not harm the cells, indicating that nuclear localization of apoptin is insufficient for its cytotoxicity. Here, we demonstrate for the first time that apoptin interacts with the SH3 domain of p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-K), through its proline-rich region. Apoptin derivatives devoid of this proline-rich region do not interact with p85, are unable to activate PI3-K, and show impaired apoptosis induction. Moreover, apoptin mutants containing the proline-rich domain are sufficient to elevate PI3-K activity and to induce apoptosis in cancer cells. Downregulation of p85 leads to nuclear exclusion of apoptin and impairs cell death induction, indicating that interaction with the p85 PI3-K subunit essentially contributes to the cytotoxic activity of apoptin. PMID:18059340

  11. IQGAP proteins reveal an atypical phosphoinositide (aPI) binding domain with a pseudo C2 domain fold.

    PubMed

    Dixon, Miles J; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R; van Aalten, Daan M F; Downes, C Peter; Batty, Ian H

    2012-06-29

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

  12. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    SciTech Connect

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H.

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  13. Signaling Pathways in Thyroid Cancer and Their Therapeutic Implications

    PubMed Central

    Jin, Shan; Borkhuu, Oyungerel; Bao, Wuyuntu; Yang, Yun-Tian

    2016-01-01

    Thyroid cancer is a common malignancy of endocrine system, and has now become the fastest increasing cancer among all the malignancies. The development, progression, invasion, and metastasis are closely associated with multiple signaling pathways and the functions of related molecules, such as Src, Janus kinase (JAK)-signal transducers and activators of transcription (STAT), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, NF-κB, thyroid stimulating hormone receptor (TSHR), Wnt-β-catenin and Notch signaling pathways. Each of the signaling pathways could exert its function singly or through network with other pathways. These pathways could cooperate, promote, antagonize, or interact with each other to form a complex network for the regulation. Dysfunction of this network could increase the development, progression, invasion, and metastasis of thyroid cancer. Inoperable thyroid cancer still has a poor prognosis. However, signaling pathway-related targeted therapies offer the hope of longer quality of meaningful life for this small group of patients. Signaling pathway-related targets provide unprecedented opportunities for further research and clinical development of novel treatment strategies for this cancer. In the present work, the advances in these signaling pathways and targeted treatments of thyroid cancer were reviewed. PMID:26985248

  14. Phosphatidylinositol 3-kinase signaling determines kidney size

    PubMed Central

    Chen, Jian-Kang; Nagai, Kojiro; Chen, Jianchun; Plieth, David; Hino, Masayo; Xu, Jinxian; Sha, Feng; Ikizler, T. Alp; Quarles, C. Chad; Threadgill, David W.; Neilson, Eric G.; Harris, Raymond C.

    2015-01-01

    Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule–specific deletion of Pten (PtenptKO) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21Cip1/WAF and p27Kip1. Administration of rapamycin to PtenptKO mice diminished hypertrophy. Proximal tubule–specific deletion of Egfr in PtenptKO mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In PtenptKO mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of PtenptKO mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX. PMID:25985273

  15. Phosphoinositide Control of Membrane Protein Function

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K.; Cui, Meng; Baki, Lia

    2015-01-01

    Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526

  16. Role of phosphoinositides at the neuronal synapse

    PubMed Central

    Frere, Samuel G.; Chang-Ileto, Belle; Di Paolo, Gilbert

    2013-01-01

    Synaptic transmission is amongst the most sophisticated and tightly controlled biological phenomena in higher eukaryotes. In the past few decades, tremendous progress has been made in our understanding of the molecular mechanisms underlying multiple facets of neurotransmission, both pre- and postsynaptically. Brought under the spotlight by pioneer studies in the areas of secretion and signal transduction, phosphoinositides and their metabolizing enzymes have been increasingly recognized as key protagonists in fundamental aspects of neurotransmission. Not surprisingly, dysregulation of phosphoinositide metabolism has also been implicated in synaptic malfunction associated with a variety of brain disorders. In the present chapter, we summarize current knowledge on the role of phosphoinositides at the neuronal synapse and highlight some of the outstanding questions in this research field. PMID:22374090

  17. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    PubMed

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling. PMID:27082310

  18. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    PubMed

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

  19. Characterization of a Tumor-Associated Activating Mutation of the p110β PI 3-Kinase

    PubMed Central

    Dbouk, Hashem A.; Khalil, Bassem D.; Wu, Haiyan; Shymanets, Aliaksei; Nürnberg, Bernd; Backer, Jonathan M.

    2013-01-01

    The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β. PMID:23734178

  20. Characterization of a tumor-associated activating mutation of the p110β PI 3-kinase.

    PubMed

    Dbouk, Hashem A; Khalil, Bassem D; Wu, Haiyan; Shymanets, Aliaksei; Nürnberg, Bernd; Backer, Jonathan M

    2013-01-01

    The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β. PMID:23734178

  1. Alcohol induced changes in phosphoinositide signaling system in rat brain

    SciTech Connect

    Pandey, S.; Piano, M.; Schwertz, D.; Davis, J.; Pandey, G. )

    1991-03-11

    Agonist-induced phosphoinositide break down functions as a signal generating system in a manner similar to the C-AMP system. In order to examine if the changes produced by chronic ethanol treatment on membrane lipid composition and metabolism effect the cellular functions of the neuron, the authors have examined the effect of chronic ethanol exposure on norepinephrine (NE) serotonin (5HT) and calcium ionophore (CI) stimulated phosphoinositide (PI) hydrolysis in rat cortical slices. Rats were maintained on liber-decarli diet alcohol and control liquid diet containing isocaloric sucrose substitute for two months. They were then sacrificed and brain was removed for determination of PI turnover. 5HT stimulated {sup 3}H- inositol monophosphate ({sup 3}H-IPI) formation was significantly lower in the cortex of alcohol treated rats as compared to control rats. However, neither CI nor NE stimulated IP1 formation was significantly different from control rats. The results thus indicate that chronic exposure to ethanol decreases 5HT induced PI breakdown in rat cortex. In order to examine if this decrease is related to a decrease in 5HT2 receptors, or decreased in coupling of receptor to the effector pathway, the authors are currently determining the number and affinity of 5HT2 receptors in alcohol treated rats.

  2. Physical Foundations of PTEN/Phosphoinositide Interaction

    NASA Astrophysics Data System (ADS)

    Gericke, Arne; Jiang, Zhiping; Redfern, Roberta E.; Kooijman, Edgar E.; Ross, Alonzo H.

    2009-03-01

    Phosphoinositides act as signaling molecules by recruiting critical effectors to specific subcellular membranes to regulate cell proliferation, apoptosis and cytoskeletal reorganization, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. PTEN is a phosphatase specific for the 3 position of the phosophoinositide ring that is deleted or mutated in many different disease states. PTEN association with membranes requires the interaction of its C2 domain with phosphatidylserine and the interaction of its N-terminal end with phosphatidylinositol-4,5-bisphophate (PI(4,5)P2). We have investigated PTEN/PI(4,5)P2 interaction and found that Lys13 is crucial for the observed binding. We also found that the presence of cholesterol enhances PTEN binding to mixed PI(4,5)P2/POPC vesicles. Fluorescence microscopy experiments utilizing GUVs yielded results consistent with enhanced phosphoinositide domain formation in the presence of cholesterol. These experiments were accompanied by zeta potential measurements and solid state MAS ^31P-NMR experiments aimed at investigating the ionization behavior of phosphoinositides.

  3. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.

    PubMed Central

    Dowler, S; Currie , R A; Campbell , D G; Deak, M; Kular, G; Downes, C P; Alessi, D R

    2000-01-01

    The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s). PMID:11001876

  4. Clinical development of phosphatidylinositol 3-kinase inhibitors for non-Hodgkin lymphoma

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is extensively explored in cancers. It functions as an important regulator of cell growth, survival and metabolism. Activation of this pathway also predicts poor prognosis in numerous human malignancies. Drugs targeting this signaling pathway have been developed and have shown preliminary clinical activity. Accumulating evidence has highlighted the important role of PI3K in non-Hodgkin lymphoma (NHL), especially in the disease initiation and progression. Therapeutic functions of PI3K inhibitors in NHL have been demonstrated both in vivo and in vitro. This review will summarize recent advances in the activation of PI3K signaling in different types of NHL and the applications of PI3K inhibitors in NHL treatment. PMID:24252186

  5. Mirror image phosphoinositides regulate autophagy

    PubMed Central

    Vicinanza, Mariella; Rubinsztein, David C

    2016-01-01

    Autophagosome formation is stimulated by canonical VPS34-dependent formation of phosphatidylinositol 3-phosphate [PI(3)P], which recruits effectors such as WIPI2. However, non-canonical VPS34-independent autophagy has also been proposed. We recently described that PI(5)P regulates autophagosome biogenesis, recruits WIPI2, and rescues autophagy in VPS34-inactivated cells. These alternative autophagy-initiating pathways reveal new druggable targets for treating neurodegeneration and cancer. PMID:27308581

  6. Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line.

    PubMed

    Rosa Santos, S C; Dumon, S; Mayeux, P; Gisselbrecht, S; Gouilleux, F

    2000-02-24

    Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression. PMID:10713704

  7. Tim-1-Mediated T Cell Activation Requires Recruitment and Activation of PI 3-Kinase

    PubMed Central

    de Souza, Anjali J.; Oak, Jean S.; Jordanhazy, Ryan; DeKruyff, Rosemarie H.; Fruman, David A.; Kane, Lawrence P.

    2009-01-01

    Ligation of the transmembrane protein Tim-1 can co-stimulate T cell activation. Agonistic antibodies to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or co-stimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in an lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI 3-kinase. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation. PMID:18453570

  8. Phosphatidylinositol 3-kinase-dependent, MEK-independent proliferation in response to CaR activation

    SciTech Connect

    Bilderback, Tim R.; Lee, Fred; Auersperg, Nelly; Rodland, Karin D.

    2002-07-02

    Although ovarian surface epithelial (OSE) cells are responsible for the majority of ovarian tumors, we know relatively little about the pathway(s) that are responsible for regulating their proliferation. We found that phosphatidylinositol 3-kinase (PI3K) is activated in OSE cells in response to elevated extracellular calcium, and the PI3K inhibitors wortmannin and LY29004 inhibited ERK activation by approximately 75%, similar to effects of the MEK2 inhibitor PD98059. However, in assays of proliferation we found that PD98059 inhibited proliferation by approximately 50%, while wortmannin inhibited greater than 90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERK activation in response to calcium. These results demonstrate that ERK activation cannot account for the full proliferative effect of elevated calcium in OSE cells, and suggest the presence of an ERK independent, PI3K dependant component in the proliferative response.

  9. Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells.

    PubMed

    Kaliman, P; Canicio, J; Shepherd, P R; Beeton, C A; Testar, X; Palacín, M; Zorzano, A

    1998-01-01

    Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme. PMID:9440811

  10. Analysis of PI3K pathway components in human cancers

    PubMed Central

    DARAGMEH, JAMILA; BARRIAH, WASEIM; SAAD, BASHAR; ZAID, HILAL

    2016-01-01

    Recent advances in genomics, proteomics, cell biology and biochemistry of tumors have revealed new pathways that are aberrantly activated in numerous cancer types. However, the enormous amount of data available in this field may mislead scientists in focused research. As cancer cell growth and progression is often dependent upon the phosphoinositide 3-kinase (PI3K)/AKT pathway, there has been extensive research into the proteins implicated in the PI3K pathway. Using data available in the Human Protein Atlas database, the current study investigated the expression of 25 key proteins that are known to be involved with PI3K pathway activation in a distinct group of 20 cancer types. These proteins are AKTIP, ARP1, BAD, GSK3A, GSK3B, MERTK-1, PIK3CA, PRR5, PSTPIP2, PTEN, FOX1, RHEB, RPS6KB1, TSC1, TP53, BCL2, CCND1, WFIKKN2, CREBBP, caspase-9, PTK2, EGFR, FAS, CDKN1A and XIAP. The analysis revealed pronounced expression of specific proteins in distinct cancer tissues, which may have the potential to serve as targets for treatments and provide insights into the molecular basis of cancer. PMID:27073576

  11. PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells

    PubMed Central

    Nile, Aaron H.; Tripathi, Ashutosh; Yuan, Peihua; Mousley, Carl J.; Suresh, Sundari; Wallace, Iain Michael; Shah, Sweety D.; Pohlhaus, Denise Teotico; Temple, Brenda; Nislow, Corey; Giaever, Guri; Tropsha, Alexander; Davis, Ronald W.; St Onge, Robert P.; Bankaitis, Vytas A.

    2013-01-01

    Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. PMID:24292071

  12. PI-3 kinase p110β: a therapeutic target in advanced prostate cancers

    PubMed Central

    Li, Benyi; Sun, Aijing; Jiang, Wencong; Thrasher, J Brantley; Terranova, Paul

    2014-01-01

    Prostate cancers in the castration-resistant stage are life-threatening because they are not curable in clinic. The novel androgen receptor inhibitor Xandi (Enzalutamide) and the new CYP17 inhibitor Zytiga (Abiraterone) prolonged patient survival only a few months in advanced prostate cancers. Therefore, novel therapeutic agents for advanced prostate cancers are urgently needed. PI-3 kinases are major intracellular signaling molecules that regulate multiple signal pathways related to cellular metabolism, cytokinesis, growth and survival. Accumulating evidence in the literature indicates that some isoforms of this kinase family are oncogenic and abnormally expressed in various human cancers, including prostate cancers. Recent extensive studies from our group and others showed that PI-3 kinase p110β is aberrantly overexpressed in advanced prostate cancers and is critical for prostate cancer development and progression as demonstrated in cell-based and animal models. Importantly, novel p110β-specific inhibitors have been developed and are currently been testing in clinical trials. In this article, we will briefly summarize recent developments in this regard. PMID:25374921

  13. Phosphatidylinositol 3-kinase inhibitors block differentiation of skeletal muscle cells.

    PubMed

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1996-08-01

    Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells. PMID:8702591

  14. Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase and Its Relationship to Alphaherpesvirus US3 Kinases

    PubMed Central

    Erazo, Angela

    2014-01-01

    The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a basophilic kinase orthologous to the US3 protein kinases found in all alphaherpesviruses. This review summarizes current information on the ORF66 kinase, and outlines apparent differences from other US3 kinases, as well as some of the conserved functions. One critical difference is the VZV ORF66 kinase targeting of the major regulatory VZV IE62 protein to control its nuclear import and assembly into the VZV virion, which is so far unprecedented in the alphaherpesviruses. However, ORF66 targets some cellular targets which are also targeted by US3 kinases of other herpesviruses, including the histone deacetylase-1 and 2 proteins, pathways that lead to changes in actin dynamics, and the targeting of substrates of protein kinase A, including the nuclear matrix protein matrin 3. PMID:20186610

  15. The roles of phosphoinositides in mammalian autophagy.

    PubMed

    Jang, Deok-Jin; Lee, Jin-A

    2016-08-01

    Autophagy is an evolutionarily conserved cellular process for lysosomal degradation, which is involved in various physiological processes within cells. Its dysfunction is associated with many human diseases, such as cancer, liver diseases, heart diseases, and infectious diseases, including neurodegenerative diseases. Autophagy involves the formation of a double-membrane bound autophagosome and the degradation of cytosolic components via its fusion and maturation with the lysosome. One of the most important steps in the process of autophagy is membrane biogenesis during autophagosome formation/maturation from different membrane sources within cells. However, there is limited knowledge regarding: (1) how the core autophagy machinery is recruited to the initial site to initiate the formation of the isolation membrane and (2) how the autophagosome matures into the functional autolysosome. Lipid supply for nucleation/elongation of the autophagosome has been proposed as one possible mechanism. Accumulating evidence suggests the important role of phosphoinositides as phospholipids, which represent key membrane-localized signals in the regulation of fundamental cellular processes, in autophagosome formation and maturation. This review focuses on how phosphoinositides influence autophagy induction or autophagosome biogenesis/maturation, because the way they are altered by autophagy might contribute to the pathogenesis of human diseases. PMID:27350551

  16. Photoreceptor phagocytosis is mediated by phosphoinositide signaling

    PubMed Central

    Mustafi, Debarshi; Kevany, Brian M.; Genoud, Christel; Bai, Xiaodong; Palczewski, Krzysztof

    2013-01-01

    Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.—Mustafi, D., Kevany, B. M., Genoud, C., Bai, X., Palczewski, K. Photoreceptor phagocytosis is mediated by phosphoinositide signaling. PMID:23913857

  17. Discovery of selective phosphatidylinositol 3-kinase inhibitors to treat hematological malignancies.

    PubMed

    Zhu, Jingyu; Hou, Tingjun; Mao, Xinliang

    2015-08-01

    The phosphatidylinositol 3-kinase (PI3K) signaling pathway is associated with chemoresistance and poor prognosis of many cancers, including hematological malignancies (HM), such as leukemia, lymphomas, and multiple myeloma (MM). Targeting PI3K is emerging as a promising strategy in the treatment of these blood cancers. Recent approval of idelalisib, a specific inhibitor of PI3Kδ, for the treatment of several types of HM, is likely to attract more interest in search for novel PI3K inhibitors. Here, we discuss classic and cutting-edge techniques and strategies to identify PI3K inhibitors for the treatment of HM. Each technique has its own strengths and limitations, and their combined application will accelerate the drug discovery process with fewer associated costs. PMID:25857437

  18. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  19. Activation of the MAPK/ERK Cell-Signaling Pathway in Uterine Smooth Muscle Cells of Women With Adenomyosis.

    PubMed

    Streuli, Isabelle; Santulli, Pietro; Chouzenoux, Sandrine; Chapron, Charles; Batteux, Frédéric

    2015-12-01

    We investigated whether the myometrium might be intrinsically different in women with adenomyosis. We studied whether the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs) and phosphoinositide 3-kinase/mammalian target of rapamycin/AKT (PI3K/mTOR/AKT) cell-signaling pathways, implicated in the pathogenesis of endometriosis, might also be activated in uterine smooth muscle cells (uSMCs) of women with adenomyosis and measured the production of reactive oxygen species (ROS), proinflammatory mediators that modulate cell proliferation and have been shown to activate the MAPK/ERK pathway in endometriosis. The uSMC cultures were derived from myometrium biopsies obtained during hysterectomy or myomectomy in women with adenomyosis and controls with leiomyoma. Proliferation of uSMCs and in vitro activation of the MAPK/ERK cell-signaling pathway were increased in women with adenomyosis compared to controls. The activation of the PI3K/mTOR/AKT pathway was not significant. The ROS production and ROS detoxification pathways were not different between uSMCs of women with adenomyosis and controls suggesting an ROS-independent activation of the MAPK/ERK pathway. Our results also provide evidence that protein kinase inhibitors and the rapanalogue temsirolimus can control proliferation of uSMCs in vitro suggesting an implication of the MAPK/ERK and the PI3K/mTOR/AKT pathways in proliferation of uSMCs in women with adenomyosis and leiomyomas. PMID:26071388

  20. Interaction of key pathways in sorafenib-treated hepatocellular carcinoma based on a PCR-array

    PubMed Central

    Liu, Yan; Wang, Ping; Li, Shijie; Yin, Linan; Shen, Haiyang; Liu, Ruibao

    2015-01-01

    This study aimed to identify the key pathways and to explore the mechanism of sorafenib in inhibiting hepatocellular carcinoma (HCC). The gene expression profile of GSE33621, including 6 sorafenib treated group and 6 control samples, was downloaded from the GEO (Gene Expression Omnibus) database. The differentially expressed genes (DEGs) in HCC samples were screened using the ΔΔCt method with the homogenized internal GAPDH. Also, the functions and pathways of DEGs were analyzed using the DAVID. Moreover, the significant pathways of DEGs that involved in HCC were analyzed based on the Latent pathway identification analysis (LPIA). A total of 44 down-regulated DEGs were selected in HCC samples. Also, there were 84 biological pathways that these 44 DEGs involved in. Also, LPIA showed that Osteoclast differentiation and hsa04664-Fc epsilon RI signaling pathway was the most significant interaction pathways. Moreover, Apoptosis, Toll-like receptor signaling pathway, Chagas disease, and T cell receptor signaling pathway were the significant pathways that interacted with hsa04664. In addition, DEGs such as AKT1 (v-akt murine thymoma viral oncogene homolog 1), TNF (tumor necrosis factor), SYK (spleen tyrosine kinase), and PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1 (alpha)) were the common genes that involved in the significant pathways. Several pathway interaction pairs that caused by several downregulated genes such as SYK, PI3K, AKT1, and TNF, were identified play curial role in sorafenib treated HCC. Sorafenib played important inhibition roles in HCC by affecting a complicate pathway interaction network. PMID:26045814

  1. Investigation of molecular mechanisms and regulatory pathways of pro-angiogenic nanorods

    NASA Astrophysics Data System (ADS)

    Nethi, Susheel Kumar; Veeriah, Vimal; Barui, Ayan Kumar; Rajendran, Saranya; Mattapally, Saidulu; Misra, Sanjay; Chatterjee, Suvro; Patra, Chitta Ranjan

    2015-05-01

    Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular mechanisms and signaling pathways in order to develop the most efficient and effective alternative treatment strategy for CVDs. However, the exact underlying mechanism and cascade signaling pathways behind the pro-angiogenic properties exhibited by EHNs still remain unclear. Herein, we report for the first time that the hydrogen peroxide (H2O2), a redox signaling molecule, generated by these EHNs activates the endothelial nitric oxide synthase (eNOS) that promotes the nitric oxide (NO) production in a PI3K (phosphoinositide 3-kinase)/Akt dependent manner, eventually triggering angiogenesis. We intensely believe that the investigation and understanding of the in-depth molecular mechanism and signaling pathways of EHNs induced angiogenesis will help us in developing an effective alternative treatment strategy for cardiovascular related and ischemic diseases where angiogenesis plays an important role.Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular

  2. Genomic Determinants of PI3K Pathway Inhibitor Response in Cancer

    PubMed Central

    Weigelt, Britta; Downward, Julian

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer as a result of genetic (e.g., amplifications, mutations, deletions) and epigenetic (e.g., methylation, regulation by non-coding RNAs) aberrations targeting its key components. Several lines of evidence demonstrate that tumors from different anatomical sites depend on the continued activation of this pathway for the maintenance of their malignant phenotype. The PI3K pathway therefore is an attractive candidate for therapeutic intervention, and inhibitors targeting different components of this pathway are in various stages of clinical development. Burgeoning data suggest that the genomic features of a given tumor determine its response to targeted small molecule inhibitors. Importantly, alterations of different components of the PI3K pathway may result in distinct types of dependencies and response to specific therapeutic agents. In this review, we will focus on the genomic determinants of response to PI3K, dual PI3K/mechanistic target of rapamycin (mTOR), mTOR, and AKT inhibitors in cancer identified in preclinical models and clinical trials to date, and the development of molecular tools for the stratification of cancer patients. PMID:22970424

  3. Phophatidylinositol-3 kinase/mammalian target of rapamycin/p70S6K regulates contractile protein accumulation in airway myocyte differentiation.

    PubMed

    Halayko, Andrew J; Kartha, Sreedharan; Stelmack, Gerald L; McConville, John; Tam, John; Camoretti-Mercado, Blanca; Forsythe, Sean M; Hershenson, Marc B; Solway, Julian

    2004-09-01

    Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells. PMID:15105162

  4. 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

  5. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    PubMed Central

    Li, Yaofeng; Chen, Xiangyun; Tang, Xiaofang; Zhang, Chundong; Wang, La; Chen, Peng; Pan, Minhui; Lu, Cheng

    2015-01-01

    Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU) labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K)/Akt, the target of rapamycin (TOR) and the extracellular signal-regulated kinase (ERK) pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells. PMID:25794286

  6. DNA synthesis during endomitosis is stimulated by insulin via the PI3K/Akt and TOR signaling pathways in the silk gland cells of Bombyx mori.

    PubMed

    Li, Yaofeng; Chen, Xiangyun; Tang, Xiaofang; Zhang, Chundong; Wang, La; Chen, Peng; Pan, Minhui; Lu, Cheng

    2015-01-01

    Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU) labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K)/Akt, the target of rapamycin (TOR) and the extracellular signal-regulated kinase (ERK) pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells. PMID:25794286

  7. Phosphoinositides and the regulation of tubular-based endosomal sorting.

    PubMed

    Cullen, Peter J

    2011-08-01

    From the pioneering work of Mabel and Lowell Hokin in the 1950s, the biology of this specific isomer of hexahydroxycyclohexane and its phosphorylated derivatives, in the form of inositol phosphates and phosphoinositides, has expanded to fill virtually every corner of cell biology, whole-organism physiology and development. In the present paper, I give a personal view of the role played by phosphoinositides in regulating the function of the endosomal network, and, in so doing, highlight some of the basic properties through which phosphoinositides regulate cell function. PMID:21787311

  8. Phosphoinositides: Important lipids in the coordination of cell dynamics.

    PubMed

    Viaud, Julien; Mansour, Rana; Antkowiak, Adrien; Mujalli, Abdulrahman; Valet, Colin; Chicanne, Gaëtan; Xuereb, Jean-Marie; Terrisse, Anne-Dominique; Séverin, Sonia; Gratacap, Marie-Pierre; Gaits-Iacovoni, Frédérique; Payrastre, Bernard

    2016-06-01

    By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field. PMID:26391221

  9. A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

    PubMed Central

    Chen, Xiao-Wei; Leto, Dara; Xiong, Tingting; Yu, Genggeng; Cheng, Alan; Decker, Stuart; Saltiel, Alan R.

    2011-01-01

    Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation. PMID:21148297

  10. Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload

    NASA Technical Reports Server (NTRS)

    Carlson, C. J.; Fan, Z.; Gordon, S. E.; Booth, F. W.

    2001-01-01

    Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.

  11. Plant phosphoinositide signaling - dynamics on demand.

    PubMed

    Heilmann, Ingo

    2016-09-01

    Eukaryotic membranes contain small amounts of lipids with regulatory roles. An important class of such regulatory lipids are phosphoinositides (PIs). Within membranes, PIs serve as recruitment signals, as regulators of membrane protein function or as precursors for second messenger production, thereby influencing a multitude of cellular processes with key importance for plant function and development. Plant PIs occur locally and transiently within membrane microdomains, and their abundance is strictly controlled. To understand the functions of the plant PI-network it is important to understand not only downstream PI-effects, but also to identify and characterize factors contributing to dynamic PI formation. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26924252

  12. The cytomegalovirus homolog of interleukin-10 requires phosphatidylinositol 3-kinase activity for inhibition of cytokine synthesis in monocytes.

    PubMed

    Spencer, Juliet V

    2007-02-01

    Human cytomegalovirus (CMV) has evolved numerous strategies for evading host immune defenses, including piracy of cellular cytokines. A viral homolog of interleukin-10, designated cmvIL-10, binds to the cellular IL-10 receptor and effects potent immune suppression. The signaling pathways employed by cmvIL-10 were investigated, and the classic IL-10R/JAK1/Stat3 pathway was found to be activated in monocytes. However, inhibition of JAK1 had little effect on cmvIL-10-mediated suppression of tumor necrosis factor alpha (TNF-alpha) production. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway had a more significant impact on TNF-alpha levels but did not completely relieve the immune suppression, demonstrating that cmvIL-10 stimulates multiple signaling pathways to modulate cell function. PMID:17121792

  13. Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

    PubMed Central

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Wei, Yan-Quan; Feng, Xia; Yao, Xue-Ping; Cao, Sui-Zhong; Xiang Liu, Ding; Liu, Xiang-Tao

    2016-01-01

    Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na+/H+ exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses. PMID:26757826

  14. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling

    PubMed Central

    Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

    2016-01-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  15. Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase.

    PubMed

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Wei, Yan-Quan; Feng, Xia; Yao, Xue-Ping; Cao, Sui-Zhong; Xiang Liu, Ding; Liu, Xiang-Tao

    2016-01-01

    Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na(+)/H(+) exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses. PMID:26757826

  16. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

    PubMed

    Cinnamon, Einat; Makki, Rami; Sawala, Annick; Wickenberg, Leah P; Blomquist, Gary J; Tittiger, Claus; Paroush, Ze'ev; Gould, Alex P

    2016-08-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  17. Modulation of neurotrophic signaling pathways by polyphenols

    PubMed Central

    Moosavi, Fatemeh; Hosseini, Razieh; Saso, Luciano; Firuzi, Omidreza

    2016-01-01

    Polyphenols are an important class of phytochemicals, and several lines of evidence have demonstrated their beneficial effects in the context of a number of pathologies including neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. In this report, we review the studies on the effects of polyphenols on neuronal survival, growth, proliferation and differentiation, and the signaling pathways involved in these neurotrophic actions. Several polyphenols including flavonoids such as baicalein, daidzein, luteolin, and nobiletin as well as nonflavonoid polyphenols such as auraptene, carnosic acid, curcuminoids, and hydroxycinnamic acid derivatives including caffeic acid phentyl ester enhance neuronal survival and promote neurite outgrowth in vitro, a hallmark of neuronal differentiation. Assessment of underlying mechanisms, especially in PC12 neuronal-like cells, reveals that direct agonistic effect on tropomyosin receptor kinase (Trk) receptors, the main receptors of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) explains the action of few polyphenols such as 7,8-dihydroxyflavone. However, several other polyphenolic compounds activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. Increased expression of neurotrophic factors in vitro and in vivo is the mechanism of neurotrophic action of flavonoids such as scutellarin, daidzein, genistein, and fisetin, while compounds like apigenin and ferulic acid increase cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation. Finally, the antioxidant activity of polyphenols reflected in the activation of Nrf2 pathway and the consequent upregulation of detoxification enzymes such as heme oxygenase-1 as well as the contribution of these effects to the neurotrophic activity have also been discussed. In conclusion, a better understanding of the neurotrophic effects of polyphenols and

  18. The antiepileptic drug valproic acid and other medium-chain fatty acids acutely reduce phosphoinositide levels independently of inositol in Dictyostelium

    PubMed Central

    Chang, Pishan; Orabi, Benoit; Deranieh, Rania M.; Dham, Manik; Hoeller, Oliver; Shimshoni, Jakob A.; Yagen, Boris; Bialer, Meir; Greenberg, Miriam L.; Walker, Matthew C.; Williams, Robin S. B.

    2012-01-01

    SUMMARY Valproic acid (VPA) is the most widely prescribed epilepsy treatment worldwide, but its mechanism of action remains unclear. Our previous work identified a previously unknown effect of VPA in reducing phosphoinositide production in the simple model Dictyostelium followed by the transfer of data to a mammalian synaptic release model. In our current study, we show that the reduction in phosphoinositide [PtdInsP (also known as PIP) and PtdInsP2 (also known as PIP2)] production caused by VPA is acute and dose dependent, and that this effect occurs independently of phosphatidylinositol 3-kinase (PI3K) activity, inositol recycling and inositol synthesis. In characterising the structural requirements for this effect, we also identify a family of medium-chain fatty acids that show increased efficacy compared with VPA. Within the group of active compounds is a little-studied group previously associated with seizure control, and analysis of two of these compounds (nonanoic acid and 4-methyloctanoic acid) shows around a threefold enhanced potency compared with VPA for protection in an in vitro acute rat seizure model. Together, our data show that VPA and a newly identified group of medium-chain fatty acids reduce phosphoinositide levels independently of inositol regulation, and suggest the reinvestigation of these compounds as treatments for epilepsy. PMID:21876211

  19. Rescue from Sexually Dimorphic Neuronal Cell Death by Estradiol and PI3 Kinase Activity.

    PubMed

    Cheng, Hui-Yun; Hung, Shin-Hui; Chu, Po-Ju

    2016-07-01

    Responses of primary hippocampal and cortical neurons derived from male and female rats to cellular stressors were studied. It is demonstrated that 17β-estradiol (E2), a potent neuroprotectant, protected the female neurons but had no effects on the male neurons from CoCl2- and glutamate-induced toxicity. Agonists of the estrogen receptor (ER) subtypes ERα and ERβ, DPN and PPT, respectively, had similar effects to E2. By contrast, effects of E2 were abolished by the ER antagonist ICI-182780, further corroborating the neuroprotective role of ERs. In male neurons, CoCl2 predominately activated the apoptosis-inducing factor (AIF)-dependent pathway and AIF translocation from the cytosol to the nucleus. In comparison, CoCl2 activated the caspase pathway and cytochrome c release in female neurons. The inhibitors of these pathways, namely DiQ for AIF and zVAD for caspase, specifically rescued CoCl2-induced cell death in male and female neurons, respectively. When zVAD and ICI-182780, and E2 were applied in combination, it was demonstrated E2 acted on the caspase pathway leading to female-specific neuroprotection. Furthermore, the PI3 kinase (PI3K) inhibitor blocked the rescue effects of DiQ and zVAD on the male and female neurons, respectively, suggesting that PI3K is a common upstream regulator for both pathways. The present study suggested that both sex-specific and nonspecific mechanisms played a role in neuronal responses to stressors and protective reagents. PMID:26369912

  20. GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs

    PubMed Central

    Hilioti, Zoe; Gallagher, Deirdre A.; Low-Nam, Shalini T.; Ramaswamy, Priya; Gajer, Pawel; Kingsbury, Tami J.; Birchwood, Christine J.; Levchenko, Andre; Cunningham, Kyle W.

    2004-01-01

    The conserved RCN family of proteins can bind and directly regulate calcineurin, a Ca2+-activated protein phosphatase involved in immunity, heart growth, muscle development, learning, and other processes. Whereas high levels of RCNs can inhibit calcineurin signaling in fungal and animal cells, RCNs can also stimulate calcineurin signaling when expressed at endogenous levels. Here we show that the stimulatory effect of yeast Rcn1 involves phosphorylation of a conservedserine residue by Mck1, a member of the GSK-3 family of protein kinases. Mutations at the GSK-3 consensus site of Rcn1 and human DSCR1/MCIP1 abolish the stimulatory effects on calcineurin signaling. RCNs may therefore oscillate between stimulatory and inhibitory forms in vivo in a manner similar to the Inhibitor-2 regulators of type 1 protein phosphatase. Computational modeling indicates a biphasic response of calcineurin to increasing RCN concentration such that protein phosphatase activity is stimulated by low concentrations of phospho-RCN and inhibited by high concentrations of phospho- or dephospho-RCN. This prediction was verified experimentally in yeast cells expressing Rcn1 or DSCR1/MCIP1 at different concentrations. Through the phosphorylation of RCNs, GSK-3 kinases can potentially contribute to a positive feedback loop involving calcineurin-dependent up-regulation of RCN expression. Such feedback may help explain the large induction of DSCR1/MCIP1 observed in brain of Down syndrome individuals. PMID:14701880

  1. Radiation-induced upregulation of telomerase activity escapes PI3-kinase inhibition in two malignant glioma cell lines

    PubMed Central

    MILLET, P.; GRANOTIER, C.; ETIENNE, O.; BOUSSIN, F.D.

    2013-01-01

    Tumor relapse after radiotherapy is a great concern in the treatment of high-grade gliomas. Inhibition of the PI3-kinase/AKT pathway is known to radiosensitize cancer cells and to delay their DNA repair after irradiation. In this study, we show that the radiosensitization of CB193 and T98G, two high-grade glioma cell lines, by the PI3K inhibitor LY294002, correlates with the induction of G1 and G2/M arrest, but is inconsistently linked to a delayed DNA double-strand break (DSBs) repair. The PI3K/AKT pathway has been shown to activate radioprotective factors such as telomerase, whose inhibition may contribute to the radiosensitization of cancer cells. However, we show that radiation upregulates telomerase activity in LY-294002-treated glioma cells as well as untreated controls, demonstrating a PI3K/AKT-independent pathway of telomerase activation. Our study suggests that radiosensitizing strategies based on PI3-kinase inhibition in high-grade gliomas may be optimized by additional treatments targeting either telomerase activity or telomere maintenance. PMID:23727752

  2. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.

    PubMed

    Clarke, Christopher J; Ohanian, Vasken; Ohanian, Jacqueline

    2007-05-01

    The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction. PMID:17208990

  3. A pivotal role of phosphatidylinositol 3-kinase in delaying of methyl jasmonate-induced leaf senescence.

    PubMed

    Liu, Jian; Zhou, Jun; Xing, Da

    2016-06-01

    Phosphatidylinositol 3-kinase (PI3K) and its product PI3P are involved in plant development and stress responses. Our recent report has suggested that down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H(+)-ATPase (V-ATPase). In vitro and in vivo experiment revealed that PI3K interact with VHA-B2. The inhibition of V-ATPase activity suppressed the vacuolar acidification and enhanced the stomatal opening, thereby accelerating MeJA-induced leaf senescence. It was shown that there is close relationship between PI3K and V-ATPase. However, the factor which initiates the PI3K-V-ATPase pathway needs further improvement, and the domain of VHA-B that binds to PI3K is still not clear enough. By using the Arabidopsis and MeJA as the research model, studies have been performed to investigate the upstream regulation of PI3K and downstream function of PI3K-V-ATPase pathway in the plant senescence. PMID:26906642

  4. Essential Role of the p110β Subunit of Phosphoinositide 3-OH Kinase in Male Fertility

    PubMed Central

    Ciraolo, Elisa; Morello, Fulvio; Hobbs, Robin M.; Wolf, Frieder; Marone, Romina; Iezzi, Manuela; Lu, Xiaoyun; Mengozzi, Giulio; Altruda, Fiorella; Sorba, Giovanni; Guan, Kaomei; Pandolfi, Pier Paolo; Wymann, Matthias P.

    2010-01-01

    Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110β develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110β-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110β is crucially needed in c-Kit–mediated spermatogonial expansion, as c-Kit–positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110β inhibitor. These data establish that activation of the p110β PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers. PMID:20053680

  5. Phosphoinositide metabolism and adrenergic receptors in astrocytes

    SciTech Connect

    Noble, E.P.; Ritchie, T.; de Vellis, J.

    1986-03-01

    Agonist-induced phosphoinositide (PI) breakdown functions as a signal generating system. Diacylglycerol, one breakdown product of phosphotidylinositol-4,5-diphosphate hydrolysis, can stimulate protein kinase C, whereas inositol triphosphate, the other product, has been proposed to be a second messenger for Ca/sup + +/ mobilization. Using purified astrocyte cultures from neonatal rat brain, the effects of adrenergic agonists and antagonists at 10/sup -5/ M were measured on PI breakdown. Astrocytes grown in culture were prelabeled with (/sup 3/H)inositol, and basal (/sup 3/H) inositol phosphate (IP/sub 1/) accumulation was measured in the presence of Li/sup +/. Epinephrine > norepinephrine (NE) were the most active stimulants of IP/sub 1/ production. The ..cap alpha../sub 1/ adrenoreceptor blockers, phentolamine and phenoxybenzamine, added alone had no effect on IP/sub 1/ production was reduced below basal levels. Propranolol partially blocked the effects of NE. Clonidine and isoproterenol, separately added, reduced IP/sub 1/ below basal levels and when added together diminished IP/sub 1/ accumulation even further. The role of adrenergic stimulation in the production of c-AMP.

  6. Validation and determination of taselisib, a β-sparing phosphoinositide 3-kinase (PI3K) inhibitor, in human plasma by LC-MS/MS.

    PubMed

    Ding, X; Faber, K; Shi, Y; McKnight, J; Dorshorst, D; Ware, J A; Dean, B

    2016-07-15

    A liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of taselisib (GDC-0032, RO5537381) concentrations in human plasma has been developed and validated to support bioanalysis of clinical samples. Solid phase extraction (SPE) was used to extract plasma samples (50μL) and the resulting samples were analyzed using reversed phase chromatography and mass spectrometry coupled with an atmospheric pressure chemical ionization interface. The mass analysis of taselisib was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range 0.400-400ng/mL using linear regression and 1/x(2) weighting. The within-run relative standard deviation (%RSD) ranged from 1.3 to 5.6%, while the between-run %RSD varied from 2.0 to 4.5% for LLOQ, low, medium, medium high and high QCs. The accuracy ranged from 94.7 to 100.3% of nominal for within-run and 96.0-99.0% of nominal for between-run for the same QCs. Extraction recovery of taselisib was between 83.8% and 92.9%. Stability of taselisib was established in human plasma for 977days at -20°C and -70°C and established in sample extracts for 96h when stored at 2 - 8°C. Stable-labeled internal standard was used to minimize matrix effects. Mean single dose pharmacokinetic parameters determined using this method for a phase I/II clinical trial were: Cmax=35.2ng/mL, AUC0-inf=1570ngh/mL, and T1/2=39.3h. PMID:27187764

  7. Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse

    PubMed Central

    Hu, Xiwen; Li, Jiangchao; Zhang, Qianqian; Zheng, Lingyun; Wang, Guang; Zhang, Xiaohan; Zhang, Jingli; Gu, Quliang; Ye, Yuxiang; Guo, Sun-Wei; Yang, Xuesong; Wang, Lijing

    2016-01-01

    Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δD910A/D910A), we show that fetuses carried by p110δD910A/D910A females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δD910A/D910A females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell. PMID:27306493

  8. Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling

    PubMed Central

    Roberson, Elle C.; Garcia, Galo; Abedin, Monika; Schurmans, Stéphane; Inoue, Takanari; Reiter, Jeremy F.

    2015-01-01

    SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling. PMID:26305592

  9. Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation.

    PubMed

    Toulany, Mahmoud; Rodemann, H Peter

    2015-12-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key cascade downstream of several protein kinases, especially membrane-bound receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) family members. Hyperactivation of the PI3K/Akt pathway is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in human solid tumors. Akt/PKB (Protein Kinase B) members are the major kinases that act downstream of PI3K, and these are involved in a variety of cellular functions, including growth, proliferation, glucose metabolism, invasion, metastasis, angiogenesis, and survival. Accumulating evidence indicates that activated Akt is one of the major predictive markers for solid tumor responsiveness to chemo/radiotherapy. DNA double-strand breaks (DNA-DSB), are the prime cause of cell death induced by ionizing radiation. Preclinical in vitro and in vivo studies have shown that constitutive activation of Akt and stress-induced activation of the PI3K/Akt pathway accelerate the repair of DNA-DSB and, consequently, lead to therapy resistance. Analyzing dysregulations of Akt, such as point mutations, gene amplification or overexpression, which results in the constitutive activation of Akt, might be of special importance in the context of radiotherapy outcomes. Such studies, as well as studies of the mechanism(s) by which activated Akt1 regulates repair of DNA-DSB, might help to identify combinations using the appropriate molecular targeting strategies with conventional radiotherapy to overcome radioresistance in solid tumors. In this review, we discuss the dysregulation of the components of upstream regulators of Akt as well as specific modifications of Akt isoforms that enhance Akt activity. Likewise, the mechanisms by which Akt interferes with repair of DNA after exposure to ionizing radiation, will be reviewed. Finally, the current status of Akt targeting in combination with radiotherapy will

  10. Investigation of molecular mechanisms and regulatory pathways of pro-angiogenic nanorods†

    PubMed Central

    Nethi, Susheel Kumar; Veeriah, Vimal; Barui, Ayan Kumar; Rajendran, Saranya; Mattapally, Saidulu; Misra, Sanjay

    2016-01-01

    Angiogenesis, a process involving the growth of new blood vessels from the pre-existing vasculature, plays a crucial role in various pathophysiological conditions. We have previously demonstrated that europium hydroxide [EuIII(OH)3] nanorods (EHNs) exhibit pro-angiogenic properties through the generation of reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK) activation. Considering the enormous implication of angiogenesis in cardiovascular diseases (CVDs) and cancer, it is essential to understand in-depth molecular mechanisms and signaling pathways in order to develop the most efficient and effective alternative treatment strategy for CVDs. However, the exact underlying mechanism and cascade signaling pathways behind the pro-angiogenic properties exhibited by EHNs still remain unclear. Herein, we report for the first time that the hydrogen peroxide (H2O2), a redox signaling molecule, generated by these EHNs activates the endothelial nitric oxide synthase (eNOS) that promotes the nitric oxide (NO) production in a PI3K (phosphoinositide 3-kinase)/Akt dependent manner, eventually triggering angiogenesis. We intensely believe that the investigation and understanding of the in-depth molecular mechanism and signaling pathways of EHNs induced angiogenesis will help us in developing an effective alternative treatment strategy for cardiovascular related and ischemic diseases where angiogenesis plays an important role. PMID:25963768

  11. Intracellular Signaling Pathways Involved in Childhood Acute Lymphoblastic Leukemia; Molecular Targets.

    PubMed

    Layton Tovar, Cristian Fabián; Mendieta Zerón, Hugo

    2016-06-01

    Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by an uncontrolled proliferation of immature lymphoid cells. ALL is the most common hematologic malignancy in early childhood, and it reaches peak incidence between the ages of 2 and 3 years. The prognosis of ALL is associated with aberrant gene expression, in addition to the presence of numerical or structural chromosomal alterations, age, race, and immunophenotype. The Relapse rate with regard to pharmacological treatment rises in childhood; thus, the expression of biomarkers associated with the activation of cell signaling pathways is crucial to establish the disease prognosis. Intracellular pathways involved in ALL are diverse, including Janus kinase/Signal transducers and transcription activators (JAK-STAT), Phosphoinositide-3-kinase-protein kinase B (PI3K-AKT), Ras mitogen-activated protein kinase (Ras-MAPK), Glycogen synthase kinase-3β (GSK-3β), Nuclear factor-kappa beta (NF-κB), and Hypoxia-inducible transcription factor 1α (HIF-1α), among others. In this review, we present several therapeutic targets, intracellular pathways, and molecular markers that are being studied extensively at present. PMID:27065575

  12. Interfering with Resistance to Smoothened Antagonists by Inhibition of the PI3K Pathway in Medulloblastoma

    PubMed Central

    Buonamici, Silvia; Williams, Juliet; Morrissey, Michael; Wang, Anlai; Guo, Ribo; Vattay, Anthony; Hsiao, Kathy; Yuan, Jing; Green, John; Ospina, Beatrice; Yu, Qunyan; Ostrom, Lance; Fordjour, Paul; Anderson, Dustin L.; Monahan, John E.; Kelleher, Joseph F.; Peukert, Stefan; Pan, Shifeng; Wu, Xu; Maira, Sauveur-Michel; Garcia-Echeverria, Carlos; Briggs, Kimberly J.; Watkins, D. Neil; Yao, Yung-mae; Lengauer, Christoph; Warmuth, Markus; Sellers, William R.; Dorsch, Marion

    2012-01-01

    Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in medulloblastoma. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for treating this disease. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed distinct resistance mechanisms. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, or more rarely point mutations in Smo led to reactivated Hh signaling and restored tumor growth. Unexpectedly, analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinositide 3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we demonstrated that the combination of NVP-LDE225 with the PI3K class I inhibitor NVP-BKM120 or the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. PMID:20881279

  13. Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

    SciTech Connect

    Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H.

    2012-09-17

    A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

  14. Subtype Specific MEK – PI3 Kinase Feedback as a Therapeutic Target in Pancreatic Adenocarcinoma

    PubMed Central

    Mirzoeva, Olga K.; Collisson, Eric A.; Schaefer, Peter M.; Hann, Byron; Hom, Yun K.; Ko, Andrew H.; Korn, W. Michael

    2013-01-01

    Mutations in the KRAS oncogene are dominant features in pancreatic adenocarcinoma (PDA). Since KRAS itself is considered “undruggable”, targeting pathways downstream of KRAS is being explored as a rational therapeutic strategy. We investigated the consequences of MEK inhibition in a large PDA cell line panel. Inhibition of MEK activated PI3 kinase in an EGFR-dependent fashion and combinations of MEK and EGFR inhibitors synergistically induced apoptosis. This combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. RNA expression analysis revealed predictors of susceptibility to the combination, including E-cadherin, HER3, and the miR200-family of micro-RNAs, while expression of the transcription factor ZEB1 was associated with resistance to the drug combination. Knock-down of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors. Thus, our findings suggest a new, subtype-specific and personalized therapeutic strategy for pancreatic cancer. PMID:23918833

  15. PI3-kinase mutation linked to insulin and growth factor resistance in vivo.

    PubMed

    Winnay, Jonathon N; Solheim, Marie H; Dirice, Ercument; Sakaguchi, Masaji; Noh, Hye-Lim; Kang, Hee Joon; Takahashi, Hirokazu; Chudasama, Kishan K; Kim, Jason K; Molven, Anders; Kahn, C Ronald; Njølstad, Pål R

    2016-04-01

    The phosphatidylinositol 3-kinase (PI3K) signaling pathway is central to the action of insulin and many growth factors. Heterozygous mutations in the gene encoding the p85α regulatory subunit of PI3K (PIK3R1) have been identified in patients with SHORT syndrome - a disorder characterized by short stature, partial lipodystrophy, and insulin resistance. Here, we evaluated whether SHORT syndrome-associated PIK3R1 mutations account for the pathophysiology that underlies the abnormalities by generating knockin mice that are heterozygous for the Pik3r1Arg649Trp mutation, which is homologous to the mutation found in the majority of affected individuals. Similar to the patients, mutant mice exhibited a reduction in body weight and length, partial lipodystrophy, and systemic insulin resistance. These derangements were associated with a reduced capacity of insulin and other growth factors to activate PI3K in liver, muscle, and fat; marked insulin resistance in liver and fat of mutation-harboring animals; and insulin resistance in vitro in cells derived from these mice. In addition, mutant mice displayed defective insulin secretion and GLP-1 action on islets in vivo and in vitro. These data demonstrate the ability of this heterozygous mutation to alter PI3K activity in vivo and the central role of PI3K in insulin/growth factor action, adipocyte function, and glucose metabolism. PMID:26974159

  16. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation.

    PubMed

    Bijur, Gautam N; Jope, Richard S

    2003-12-01

    We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria. PMID:14713298

  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 decreased FoxM1-induced cell proliferation. Moreover, PDGF/AKT pathway upregulates the expression of FoxM1 in breast cancer cells. Knockdown of PDGF-A or blockade of AKT activation inhibited the expression of FoxM1 in breast cancer cells. Furthermore, expression of FoxM1 significantly correlated with the expression of PDGF-A and the activated AKT signaling pathway in human breast cancer specimens. Our study demonstrates a novel positive regulatory feedback loop between FoxM1 and the PDGF/AKT signaling pathway; this loop contributes to breast cancer cell growth and tumorigenesis. PMID:25869208

  18. Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

    PubMed

    Busse, Ricarda A; Scacioc, Andreea; Schalk, Amanda M; Krick, Roswitha; Thumm, Michael; Kühnel, Karin

    2016-01-01

    Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method. PMID:26552682

  19. Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

    PubMed

    Martz, Colin A; Ottina, Kathleen A; Singleton, Katherine R; Jasper, Jeff S; Wardell, Suzanne E; Peraza-Penton, Ashley; Anderson, Grace R; Winter, Peter S; Wang, Tim; Alley, Holly M; Kwong, Lawrence N; Cooper, Zachary A; Tetzlaff, Michael; Chen, Pei-Ling; Rathmell, Jeffrey C; Flaherty, Keith T; Wargo, Jennifer A; McDonnell, Donald P; Sabatini, David M; Wood, Kris C

    2014-12-23

    Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts. PMID:25538079

  20. Systematic identification of signaling pathways with potential to confer anticancer drug resistance

    PubMed Central

    Martz, Colin A.; Ottina, Kathleen A.; Singleton, Katherine R.; Jasper, Jeff S.; Wardell, Suzanne E.; Peraza-Penton, Ashley; Anderson, Grace R.; Winter, Peter S.; Wang, Tim; Alley, Holly M.; Kwong, Lawrence N.; Cooper, Zachary A.; Tetzlaff, Michael; Chen, Pei-Ling; Rathmell, Jeffrey C.; Flaherty, Keith T.; Wargo, Jennifer A.; McDonnell, Donald P.; Sabatini, David M.; Wood, Kris C.

    2015-01-01

    Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant cDNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS– MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)–mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially-passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAFV600E melanoma cells in culture, and the abundance of Notch1 pathway markers were increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts. PMID:25538079

  1. Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

    PubMed

    Law, Nathan C; Hunzicker-Dunn, Mary E

    2016-02-26

    The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. PMID:26702053

  2. The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    PubMed Central

    Gu, Changkyu; Park, Soochul

    2001-01-01

    Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

  3. Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin.

    PubMed

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1995-12-01

    In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment. PMID:8526858

  4. Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin.

    PubMed Central

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8526858

  5. Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism

    PubMed Central

    Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

    2005-01-01

    Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase. PMID:15845472

  6. Phosphatidylinositol 3-Kinase: A Link Between Inflammation and Pancreatic Cancer.

    PubMed

    Birtolo, Chiara; Go, Vay Liang W; Ptasznik, Andrzej; Eibl, Guido; Pandol, Stephen J

    2016-01-01

    Even though a strong association between inflammation and cancer has been widely accepted, the underlying precise molecular mechanisms are still largely unknown. A complex signaling network between tumor and stromal cells is responsible for the infiltration of inflammatory cells into the cancer microenvironment. Tumor stromal cells such as pancreatic stellate cells (PSCs) and immune cells create a microenvironment that protects cancer cells through a complex interaction, ultimately facilitating their local proliferation and their migration to different sites. Furthermore, PSCs have multiple functions related to local immunity, angiogenesis, inflammation, and fibrosis. Recently, many studies have shown that members of the phosphoinositol-3-phosphate kinase (PI3K) family are activated in tumor cells, PSCs, and tumor-infiltrating inflammatory cells to promote cancer growth. Proinflammatory cytokines and chemokines secreted by immune cells and fibroblasts within the tumor environment can activate the PI3K pathway both in cancer and inflammatory cells. In this review, we focus on the central role of the PI3K pathway in regulating the cross talk between immune/stromal cells and cancer cells. Understanding the role of the PI3K pathway in the development of chronic pancreatitis and cancer is crucial for the discovery of novel and efficacious treatment options. PMID:26658038

  7. Constitutive activation of Stat5 promotes its cytoplasmic localization and association with PI3-kinase in myeloid leukemias.

    PubMed

    Harir, Noria; Pecquet, Christian; Kerenyi, Marc; Sonneck, Karoline; Kovacic, Boris; Nyga, Remy; Brevet, Marie; Dhennin, Isabelle; Gouilleux-Gruart, Valerie; Beug, Hartmut; Valent, Peter; Lassoued, Kaiss; Moriggl, Richard; Gouilleux, Fabrice

    2007-02-15

    Persistent activation of Stat5 is frequently found in hematologic neoplasms. Studies conducted with constitutively active Stat5 mutants (Stat51*6 and cS5F) have shown that deregulated Stat5 activity promotes leukemogenesis. To investigate the oncogenic properties of these mutants, we used cS5F-expressing bone marrow cells which induce a multilineage leukemia when transplanted into recipient mice. Here, we show by immunocytochemistry that cS5F is localized mainly in the cytoplasmic compartment of leukemic cells, suggesting that the transforming nature of cS5F may be associated with a cytoplasmic function. In support of this hypothesis, we found that cS5F forms a complex with the p85 subunit of the phosphatidylinositol 3-kinase (PI3-K) and the scaffolding adapter Gab2 in leukemic bone marrow cells, resulting in the activation of Akt/PKB, a crucial downstream target of PI3-K. By using transducible TAT-Gab2 or TAT-Akt recombinant proteins, we were able to demonstrate that activation of the PI3-kinase/Akt pathway by cS5F molecules through Gab2 is essential for induction of cell growth. We also found that persistently phosphorylated Stat5 in primary cells from patients with myeloid leukemias has a cytoplasmic localization. These data suggest that oncogenic Stat5 proteins exert dual transforming capabilities not only as transcriptional activators but also as cytoplasmic signaling effectors. PMID:17038539

  8. Hepatocyte growth factor (HGF) enhances cardiac commitment of differentiating embryonic stem cells by activating PI3 kinase

    SciTech Connect

    Roggia, Cristiana; Ukena, Christian; Boehm, Michael; Kilter, Heiko . E-mail: kilter@med-in.uni-saarland.de

    2007-03-10

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine promoting proliferation, migration and survival in several cell types. HGF and its cognate receptor c-Met are expressed in cardiac cells during early cardiogenesis, but data concerning its role in cardiac differentiation of embryonic stem cells (ESCs) and the underlying molecular mechanisms involved are limited. In the present study we show that HGF significantly increases the number of beating embryoid bodies of differentiating ESCs without affecting beating frequency. Furthermore, HGF up-regulates the expression of the cardiac-specific transcription factors Nkx 2.5 and GATA-4 and of markers of differentiated cardiomyocytes, i.e. {alpha}-MHC, {beta}-MHC, ANF, MLC2v and Troponin T. The HGF-induced increase in Nkx 2.5 expression was inhibited by co-treatment with the PI3 kinase inhibitors Wortmannin and LY294002, but not by its inactive homolog LY303511, suggesting an involvement of the PI3 kinase/Akt pathway in this effect. We conclude that HGF is an important growth factor involved in cardiac differentiation and/or proliferation of ESCs and may therefore be critical for the in vitro generation of pre- or fully differentiated cardiomyocytes as required for clinical use of embryonic stem cells in cardiac diseases.

  9. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading.

    PubMed

    Lakkakorpi, P T; Wesolowski, G; Zimolo, Z; Rodan, G A; Rodan, S B

    1997-12-15

    Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts. PMID:9434625

  10. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

  11. The PI3-Kinase Delta Inhibitor Idelalisib (GS-1101) Targets Integrin-Mediated Adhesion of Chronic Lymphocytic Leukemia (CLL) Cell to Endothelial and Marrow Stromal Cells

    PubMed Central

    Fiorcari, Stefania; Brown, Wells S.; McIntyre, Bradley W.; Estrov, Zeev; Maffei, Rossana; O’Brien, Susan; Sivina, Mariela; Hoellenriegel, Julia; Wierda, William G.; Keating, Michael J.; Ding, Wei; Kay, Neil E.; Lannutti, Brian J.; Marasca, Roberto; Burger, Jan A.

    2013-01-01

    CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood. PMID:24376763

  12. Pelota Regulates Epidermal Differentiation by Modulating BMP and PI3K/AKT Signaling Pathways.

    PubMed

    Elkenani, Manar; Nyamsuren, Gunsmaa; Raju, Priyadharsini; Liakath-Ali, Kifayathullah; Hamdaoui, Aicha; Kata, Aleksandra; Dressel, Ralf; Klonisch, Thomas; Watt, Fiona M; Engel, Wolfgang; Thliveris, James A; Krishna Pantakani, D V; Adham, Ibrahim M

    2016-08-01

    The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation. PMID:27164299

  13. Fragile Histidine Triad (FHIT) Suppresses Proliferation and Promotes Apoptosis in Cholangiocarcinoma Cells by Blocking PI3K-Akt Pathway

    PubMed Central

    Huang, Qiang; Liu, Zhen; Xie, Fang; Liu, Chenhai; Shao, Feng; Zhu, Cheng-lin; Hu, Sanyuan

    2014-01-01

    Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3. PMID:24757411

  14. Roles of the PI3K/Akt pathway in Epstein-Barr virus-induced cancers and therapeutic implications.

    PubMed

    Chen, Jiezhong

    2012-12-12

    Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus (EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins (LMP) 1 and LMP2A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers. PMID:24175221

  15. GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    PubMed Central

    Kim, Sang-Hoon; Kim, Hyun-Jung; Kim, Chan-Wha

    2016-01-01

    Podocyte foot processes are interdigitated to form the slit diaphragm and are crucial for the glomerular filtration barrier. Glucocorticoid-induced transcript 1 (GLCCI1) is transcriptionally regulated, but its signaling pathway in podocytes is unknown. The main objective of this study was to investigate the regulation of podocyte foot process proteins and to investigate the role of GLCCI1 in the phosphoinositide 3-kinase (PI3K) pathway using high glucose-induced podocytes and streptozotocin-induced diabetic rats. In podocytes and rat kidneys, GLCCI1 was found to be highly specific for the glomerulus and podocyte foot processes similar to other podocyte-specific proteins (nephrin, podocin, synatopodin and podocalyxin) based on reverse transcription-PCR, western blotting, immunofluorescence and immunoelectron microscopy analyses. In addition, the decrease in the GLCCI1 expression level under hyperglycemic conditions was restored by treatment with a PI3K inhibitor (wortmannin). Immunofluorescence analysis confirmed that GLCCI1 colocalized with nephrin and synaptopodin both in vivo and in vitro. Finally, immunoelectron microscopy data from streptozotocin-induced diabetic rats showed that GLCCI1 also localized in podocyte foot processes. Hence, GLCCI1 is a component of podocyte foot processes, and its expression appears to be regulated via the PI3K pathway. PMID:27174202

  16. PI3K/Akt signaling pathway is involved in the neurotrophic effect of senegenin.

    PubMed

    Pi, Ting; Zhou, Xiao-Wen; Cai, Liang; Zhang, Wei; Su, Chao-Fen; Wu, Wu-Tian; Ren, Xiao-Ming; Luo, Huan-Min

    2016-02-01

    Neurodegenerative diseases are frequently associated with the loss of synapses and neurons. Senegenin, extracted from the Chinese herb Polygala tenuifolia Willd, was previously found to promote neurite outgrowth and neuronal survival in primary cultured rat cortical neurons. The aim of the present study was to investigate the underlying mechanisms of senegenin-induced neurotrophic effects on rat cortical neurons. Primary cortical rat neurons were treated with various pharmacological antagonists and with or without senegenin, and subjected to MTT and western blot analysis to explore the effects of senegenin on cell survival as well as the activation of signaling pathways. Neurite outgrowth and neuronal survival induced by senegenin were significantly inhibited by A2A receptor antagonist ZM241385 and specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, but not by tropomyosin receptor kinase A receptor inhibitor K252a, mitogen-activated protein kinase kinase inhibitor PD98059 or protein kinase C inhibitor GÖ6976. Furthermore, senegenin enhanced the phosphorylation of Akt, which was blocked by LY294002. The present study revealed that the PI3K/Akt signaling pathway may be involved in the neurotrophic effects of senegenin. PMID:26647727

  17. Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

    PubMed Central

    Kruse, Martin; Vivas, Oscar; Traynor-Kaplan, Alexis

    2016-01-01

    In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons. PMID:26818524

  18. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    SciTech Connect

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  19. Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    PubMed Central

    Shugg, Ryan P. P.; Thomson, Ashley; Tanabe, Natsuko; Kashishian, Adam; Steiner, Bart H.; Puri, Kamal D.; Pereverzev, Alexey; Lannutti, Brian J.; Jirik, Frank R.; Dixon, S. Jeffrey; Sims, Stephen M.

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics. PMID:24133210

  20. Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

    PubMed Central

    Wang, Kelong; Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.

    2012-01-01

    Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18 to 10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and

  1. 3-Phosphoinositide-Dependent protein Kinase-1 (PDK1) inhibitors: A review from patent literature

    PubMed Central

    Barile, Elisa; De, Surya K.; Pellecchia, Maurizio

    2016-01-01

    PDK1 (3-Phosphoinositide-dependent kinase 1) is a key member of the AGC protein kinase family. It plays an important role in a variety of cellular functions, leading to the activation of the PI3K signaling pathway, an event often associated with the onset and progression of several human cancers. Numerous recent observations suggest that PDK1 inhibitors may provide novel opportunities for the development of effective classes of therapeutics. On these premises, recent years have witnessed an increased effort by medicinal chemists to develop novel scaffolds to derive potent and selective PDK1 inhibitors. The intent of this review is to update the reader on the recent patent literature covering applications published between June 2008 and September 2011 that report on PDK1 inhibitors. PMID:24236780

  2. Activation of phosphatidylinositol 3-kinase/Akt signaling mediates sorafenib-induced invasion and metastasis in hepatocellular carcinoma.

    PubMed

    Wang, Haiyong; Xu, Litao; Zhu, Xiaoyan; Wang, Peng; Chi, Huiying; Meng, Zhiqiang

    2014-10-01

    Sorafenib, an antiangiogenic agent, can promote tumor invasion and metastasis. The phosphatidylinositol 3-kinase (PI3K)/Akt/Snail-dependent pathway plays an important role in tumor invasion and metastasis. Yet, little is known concerning the role of the PI3K/Akt/Snail-dependent pathway in sorafenib‑induced invasion and metastasis of hepatic carcinoma (HCC). A human HCC orthotopic xenograft model was established, and sorafenib (30 mg/kg/day) was administered orally. Tumor growth and intrahepatic metastasis were assessed, and immunohistochemistry was applied to analyze the activation of the PI3K/Akt/Snail-dependent pathway. HCC cell lines were treated with sorafenib (1, 5 and 10 µM), and proliferation, migration and invasion were assessed. Western blotting and real-time polymerase chain reaction (RT-PCR) were used to examine the related gene expression of epithelial-mesenchymal transition (EMT) markers and the PI3K/Akt/Snail-dependent pathway. Sorafenib inhibited tumor growth and promoted intrahepatic invasion and metastasis of the orthotopic tumors grown from SMMC7721-GFP cells in vivo. Additionally, sorafenib promoted EMT and invasion and metastasis of HCC cells in vitro. Importantly, sorafenib enhanced PI3K and Akt activation and upregulation of the expression of transcription factor Snail, a critical EMT mediator. The upregulation of transcription factor Snail expression by sorafenib may be related to activation of the PI3K/AKT signaling pathway. The PI3K/Akt/Snail-dependent pathway may mediate the pro-invasive and pro-metastatic effects of sorafenib on HCC by inducing EMT. PMID:25070581

  3. α-2,8-Sialyltransferase Is Involved in the Development of Multidrug Resistance via PI3K/Akt Pathway in Human Chronic Myeloid Leukemia.

    PubMed

    Zhang, Xu; Dong, Weijie; Zhou, Huimin; Li, Hongshuai; Wang, Ning; Miao, Xiaoyan; Jia, Li

    2015-02-01

    Cell surface sialylation is emerging as an important feature of cancer cell multidrug resistance (MDR). We have focused on the influence of 2,8-sialyltransferases in key steps of the development of MDR in chronic myeloid leukemia (CML). The expressional profiles of six α-2,8-sialyltransferases were generated in three pairs of CML cell lines and peripheral blood mononuclear cells (PBMC) of CML patients. Cellular MDR phenotype positively correlated with ST8SIA4 and ST8SIA6 levels. Furthermore, ST8SIA4 mediated the activity of phosphoinositide-3 kinase (PI3K)/Akt signal pathway and the expression of P-glycoprotein (P-gp). Targeting the PI3K/Akt pathway by its specific inhibitor LY294002, or by Akt RNA interfering reversed the MDR phenotype of K562/ADR cells. Inhibition of PI3K/Akt pathway also attenuated the effects caused by the overexpression of ST8SIA4 on MDR. Therefore this study indicated that α-2,8-sialyltransferases involved in the development of MDR of CML cells probably through ST8SIA4 regulating the activity of PI3K/Akt signaling and the expression of P-gp. PMID:25855199

  4. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    PubMed

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

    2016-06-01

    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  5. Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation.

    PubMed

    Shinozaki-Narikawa, Naeko; Kodama, Tatsuhiko; Shibasaki, Yoshikazu

    2006-11-01

    Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics. PMID:17010122

  6. Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

    PubMed Central

    2013-01-01

    Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease. PMID:23899561

  7. MEF2 Is a Converging Hub for Histone Deacetylase 4 and Phosphatidylinositol 3-Kinase/Akt-Induced Transformation

    PubMed Central

    Di Giorgio, Eros; Clocchiatti, Andrea; Piccinin, Sara; Sgorbissa, Andrea; Viviani, Giulia; Peruzzo, Paolo; Romeo, Salvatore; Rossi, Sabrina; Dei Tos, Angelo Paolo; Maestro, Roberta

    2013-01-01

    The MEF2-class IIa histone deacetylase (HDAC) axis operates in several differentiation pathways and in numerous adaptive responses. We show here that nuclear active HDAC4 and HDAC7 display transforming capability. HDAC4 oncogenic potential depends on the repression of a limited set of genes, most of which are MEF2 targets. Genes verified as targets of the MEF2-HDAC axis are also under the influence of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that affects MEF2 protein stability. A signature of MEF2 target genes identified by this study is recurrently repressed in soft tissue sarcomas. Correlation studies depicted two distinct groups of soft tissue sarcomas: one in which MEF2 repression correlates with PTEN downregulation and a second group in which MEF2 repression correlates with HDAC4 levels. Finally, simultaneous pharmacological inhibition of the PI3K/Akt pathway and of MEF2-HDAC interaction shows additive effects on the transcription of MEF2 target genes and on sarcoma cells proliferation. Overall, our work pinpoints an important role of the MEF2-HDAC class IIa axis in tumorigenesis. PMID:24043307

  8. Acetylcholinesterase inhibitors used in treatment of Alzheimer's disease prevent glutamate neurotoxicity via nicotinic acetylcholine receptors and phosphatidylinositol 3-kinase cascade.

    PubMed

    Takada-Takatori, Yuki; Kume, Toshiaki; Sugimoto, Mitsuhiro; Katsuki, Hiroshi; Sugimoto, Hachiro; Akaike, Akinori

    2006-09-01

    We show here that donepezil, galanathamine and tacrine, therapeutic acetylcholinesterase inhibitors currently being used for treatment of Alzheimer's disease, protect neuronal cells in a time- and concentration-dependent manner from glutamate neurotoxicity that involves apoptosis. The neuroprotective effects were antagonized by mecamylamine, an inhibitor of nicotinic acetylcholine receptors (nAChRs). Dihydro-beta-erythroidine and methyllycaconitine, antagonists for alpha4-nAChR and alpha7-nAChR, respectively, antagonized the protective effect of donepezil and galanthamine, but not that of tacrine. Previous reports suggest the involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in the nicotine-induced neuroprotection. Inhibitors for a non-receptor type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. Furthermore, LY294002, a PI3K inhibitor, also suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galanthamine treatment, but not with tacrine treatment. These results suggest that donepezil and galanthamine prevent glutamate neurotoxicity through alpha4- and alpha7-nAChRs, followed by the PI3K-Akt pathway, and that tacrine protects neuronal cells through a different pathway. PMID:16762377

  9. Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells.

    PubMed

    Lee, Seung Hwan; Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won-Sup; Kim, Eun-Hee; Park, Hyeon Soo; Kim, Gon Sup

    2015-12-01

    Korean Citrus aurantium L. has long been used as a medicinal herb for its anti-inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose-dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide-3-kinase/Akt pathway - P-4EBP1 and P-p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl-2 and Bcl-xL were decreased with an increase in the expression of Bax/Bcl-xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid-treated HepG2 cells. It was also observed that the P-p38 protein level was increased both dose and time dependently in flavonoid-treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer. PMID:26439681

  10. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

    PubMed

    Daulhac, L; Kowalski-Chauvel, A; Pradayrol, L; Vaysse, N; Seva, C

    1999-07-16

    We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. PMID:10400698

  11. Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages*

    PubMed Central

    Välimäki, Elina; Miettinen, Juho J.; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A.

    2013-01-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  12. PI3K at the crossroads of tumor angiogenesis signaling pathways

    PubMed Central

    Soler, Adriana; Angulo-Urarte, Ana; Graupera, Mariona

    2015-01-01

    Tumors need blood vessels for their growth, thus providing the rationale for antiangiogenic therapy in cancer treatment. However, intrinsic and acquired resistance and low response rates have turned out to be major limitations of antiangiogenic therapy. This emphasizes the need to further understand how the vasculature in cancer can be targeted. Although endothelial cells (ECs) rely on multiple growth factors and cytokines to grow, antiangiogenic therapies have mainly centered on targeting vascular endothelial growth factor (VEGF). Phosphoinositide 3-kinases (PI3Ks) form a family of 8 isoenzymes with non-redundant functions in normal biology and cancer. The subgroup of class I PI3Ks are situated at the crossroad of a plethora of proangiogenic signals and control cell growth, survival, motility, and metabolism. These isoenzymes have pleiotropic roles in the tumor microenvironment, including cell-autonomous functions in ECs, underscoring the complexity of targeting this pathway in cancer. Here, we describe how the PI3K axis influences angiogenesis in different cell compartments and summarize the diversity of vascular responses to PI3K inhibition. Targeting PI3K signaling by isoform-selective inhibitors, together with readjusting the current doses below the maximum tolerated dose, may improve clinical responses to class I PI3K anticancer agents. PMID:27308431

  13. Autism as early neurodevelopmental disorder: evidence for an sAPPα-mediated anabolic pathway

    PubMed Central

    Lahiri, Debomoy K.; Sokol, Deborah K.; Erickson, Craig; Ray, Balmiki; Ho, Chang Y.; Maloney, Bryan

    2013-01-01

    Autism is a neurodevelopmental disorder marked by social skills and communication deficits and interfering repetitive behavior. Intellectual disability often accompanies autism. In addition to behavioral deficits, autism is characterized by neuropathology and brain overgrowth. Increased intracranial volume often accompanies this brain growth. We have found that the Alzheimer’s disease (AD) associated amyloid-β precursor protein (APP), especially its neuroprotective processing product, secreted APP α, is elevated in persons with autism. This has led to the “anabolic hypothesis” of autism etiology, in which neuronal overgrowth in the brain results in interneuronal misconnections that may underlie multiple autism symptoms. We review the contribution of research in brain volume and of APP to the anabolic hypothesis, and relate APP to other proteins and pathways that have already been directly associated with autism, such as fragile X mental retardation protein, Ras small GTPase/extracellular signal-regulated kinase, and phosphoinositide 3 kinase/protein kinase B/mammalian target of rapamycin. We also present additional evidence of magnetic resonance imaging intracranial measurements in favor of the anabolic hypothesis. Finally, since it appears that APP’s involvement in autism is part of a multi-partner network, we extend this concept into the inherently interactive realm of epigenetics. We speculate that the underlying molecular abnormalities that influence APP’s contribution to autism are epigenetic markers overlaid onto potentially vulnerable gene sequences due to environmental influence. PMID:23801940

  14. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  15. Phosphoinositide-signaling is one component of a robust plant defense response.

    PubMed

    Hung, Chiu-Yueh; Aspesi, Peter; Hunter, Melissa R; Lomax, Aaron W; Perera, Imara Y

    2014-01-01

    The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca(2+)-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca(2+) release, modulates defense gene expression and compromises plant defense responses. PMID:24966862

  16. Phosphoinositide-signaling is one component of a robust plant defense response

    PubMed Central

    Hung, Chiu-Yueh; Aspesi Jr, Peter; Hunter, Melissa R.; Lomax, Aaron W.; Perera, Imara Y.

    2014-01-01

    The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca2+-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca2+ release, modulates defense gene expression and compromises plant defense responses. PMID:24966862

  17. The role of inositol 1,4,5-trisphosphate 3-kinase A in regulating emotional behavior and amygdala function

    PubMed Central

    Chung, Sooyoung; Kim, Il Hwan; Lee, Dongmin; Park, Kyungjoon; Kim, Joo Yeon; Lee, Yeon Kyung; Kim, Eun Joo; Lee, Hyun Woo; Choi, June-seek; Son, Gi Hoon; Sun, Woong; Shin, Ki Soon; Kim, Hyun

    2016-01-01

    Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) is a molecule enriched in the brain and neurons that regulates intracellular calcium levels via signaling through the inositol trisphosphate receptor. In the present study, we found that IP3K-A expression is highly enriched in the central nucleus of the amygdala (CeA), which plays a pivotal role in the processing and expression of emotional phenotypes in mammals. Genetic abrogation of IP3K-A altered amygdala gene expression, particularly in genes involved in key intracellular signaling pathways and genes mediating fear- and anxiety-related behaviors. In agreement with the changes in amygdala gene expression profiles, IP3K-A knockout (KO) mice displayed more robust responses to aversive stimuli and spent less time in the open arms of the elevated plus maze, indicating high levels of innate fear and anxiety. In addition to behavioral phenotypes, decreased excitatory and inhibitory postsynaptic current and reduced c-Fos immunoreactivity in the CeA of IP3K-A KO mice suggest that IP3K-A has a profound influence on the basal activities of fear- and anxiety-mediating amygdala circuitry. In conclusion, our findings collectively demonstrate that IP3K-A plays an important role in regulating affective states by modulating metabotropic receptor signaling pathways and neural activity in the amygdala. PMID:27053114

  18. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis

    PubMed Central

    Button, Robert W.; Vincent, Joseph H.; Strang, Conor J.; Luo, Shouqing

    2016-01-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy. PMID:26814436

  19. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  20. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

    PubMed

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W-Y; Puga, Alvaro; Xia, Ying

    2015-08-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  1. Ethanol effects on rat brain phosphoinositide metabolism

    SciTech Connect

    Huang, H.M.

    1987-01-01

    An increase in acidic phospholipids in brain plasma and synaptic plasma membranes upon chronic ethanol administration was observed. Chronic ethanol administration resulted in an increase in {sup 32}P{sub i} incorporation into the acidic phospholipids in synaptosomes. Postdecapitative ischemic treatment resulted rapid degradation of poly-PI in rat brain. However, there was a rapid appearance of IP{sub 2} in ethanol group which indicated a more rapid turnover of IP{sub 3} in the ethanol-treated rats. Carbachol stimulated accumulation of labeled inositol phosphates in brain slices and synaptosomes. Carbachol-stimulated release of IP and IP{sub 2} was calcium dependent and was inhibited by EGTA and atropine. Adenosine triphosphates and 1 mM further enhanced carbachol-induced formation of IP and IP{sub 2}, but showed an increase and a decrease in IP{sub 3} at 1 mM and 0.01 mM, respectively. Guanosine triphosphate at 0.1 mM did not change in labeled IP, but there was a significant increase in labeled IP{sub 2} and decrease in IP{sub 3}. Mn and CMP greatly enhanced incorporation of ({sup 3}H)-inositol into PI, but not into poly-PI labeling in brain synaptosomes. Incubation of brain synaptosomes resulted in a Ca{sup 2+}, time-dependent release of labeled IP. However, the pool of PI labeled through this pathway is not susceptible to carbachol stimulation. When saponin permeabilized synaptosomal preparations were incubated with ({sup 3}H)-inositol-PI or ({sup 14}C)-arachidonoyl-PI, ATP enhanced the formation of labeled IP and DG.

  2. Two Pathways through Cdc42 Couple the N-Formyl Receptor to Actin Nucleation in Permeabilized Human Neutrophils

    PubMed Central

    Glogauer, M.; Hartwig, J.; Stossel, T.

    2000-01-01

    We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends. PMID:10953003

  3. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

    PubMed Central

    Jiang, Bing-Hua; Aoki, Masahiro; Zheng, Jenny Z.; Li, Jian; Vogt, Peter K.

    1999-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation. PMID:10051597

  4. PI3K/Akt pathway restricts epithelial adhesion of Dr+ Escherichia coli by down-regulating the expression of Decay Accelerating Factor (DAF)

    PubMed Central

    Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel; Nowicki, Bogdan J.; Nowicki, Stella; Yallampalli, Chandra

    2014-01-01

    The urogenital microbial infection in pregnancy is an important cause of maternal and neonatal morbidity and mortality. Uropathogenic Escherichia coli strains which express Dr fimbriae (Dr+) are associated with unique gestational virulence and they utilize cell surface decay accelerating factor (DAF or CD55) as one of the cellular receptor before invading the epithelial cells. Previous studies in our laboratory established that nitric oxide reduces the rate of E. coli invasion by delocalizing the DAF protein from cell surface lipid rafts and down-regulating its expression. The phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) cell signal pathway plays an important role in host-microbe interaction because many bacteria including E. coli activate this pathway in order to establish infection. In the present study we showed that the PI3K/Akt pathway negatively regulates the expression of DAF on the epithelial cell surface and thus inhibits the adhesion of Dr+ E. coli to epithelial cells. Initially, using two human cell lines Ishikawa and HeLa which differ in constitutive activity of PI3K/Akt we showed that DAF levels were associated with the PI3K/Akt pathway. We then showed that the DAF gene expression was up-regulated and the Dr+ E. coli adhesion increased after the suppression of PI3K/Akt pathway in Ishikawa cells using inhibitor LY-294002, and a plasmid which allowed the expression of PI3K/Akt regulatory protein PTEN. The down-regulation of PTEN protein using PTEN-specific siRNA activated the PI3K/Akt pathway, down-regulated the DAF and decreased the adhesion of Dr+ E. coli. We conclude that the PI3K/Akt pathway regulated the DAF expression in a nitric oxide independent manner. PMID:24599886

  5. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  6. Identification of differential PI3K pathway target dependencies in T-cell acute lymphoblastic leukemia through a large cancer cell panel screen.

    PubMed

    Lynch, James T; McEwen, Robert; Crafter, Claire; McDermott, Ultan; Garnett, Mathew J; Barry, Simon T; Davies, Barry R

    2016-04-19

    Selective phosphoinositide 3-kinase (PI3K)/AKT/mTOR inhibitors are currently under evaluation in clinical studies. To identify tumor types that are sensitive to PI3K pathway inhibitors we screened compounds targeting PI3Kα/δ (AZD8835), PI3Kβ/δ (AZD8186), AKT (AZD5363) and mTORC1/2 (AZD2014) against a cancer cell line panel (971 cell lines). There was an enrichment of hematological malignancies that were sensitive to AKT and mTOR inhibition, with the greatest degree of sensitivity observed in T-cell acute lymphoblastic leukemia (T-ALL). We found that all NOTCH mutant T-ALL cell lines were sensitive to AKT and mTORC1/2 inhibitors, with only partial sensitivity to agents that target the PI3K α, β or δ isoforms. Induction of apoptosis only occurred following AKTi treatment in cell lines with PTEN protein loss and high levels of active AKT. In summary, we have demonstrated that T-ALL cell lines show differential sensitivity to inhibition at different nodes in the PI3K/AKT/mTOR pathway and inhibiting AKT or mTOR may have a therapeutic benefit in this disease setting. PMID:26989080

  7. Identification of differential PI3K pathway target dependencies in T-cell acute lymphoblastic leukemia through a large cancer cell panel screen

    PubMed Central

    Lynch, James T.; McEwen, Robert; Crafter, Claire; McDermott, Ultan; Garnett, Mathew J.; Barry, Simon T.; Davies, Barry R.

    2016-01-01

    Selective phosphoinositide 3-kinase (PI3K)/AKT/mTOR inhibitors are currently under evaluation in clinical studies. To identify tumor types that are sensitive to PI3K pathway inhibitors we screened compounds targeting PI3Kα/δ (AZD8835), PI3Kβ/δ (AZD8186), AKT (AZD5363) and mTORC1/2 (AZD2014) against a cancer cell line panel (971 cell lines). There was an enrichment of hematological malignancies that were sensitive to AKT and mTOR inhibition, with the greatest degree of sensitivity observed in T-cell acute lymphoblastic leukemia (T-ALL). We found that all NOTCH mutant T-ALL cell lines were sensitive to AKT and mTORC1/2 inhibitors, with only partial sensitivity to agents that target the PI3K α, β or δ isoforms. Induction of apoptosis only occurred following AKTi treatment in cell lines with PTEN protein loss and high levels of active AKT. In summary, we have demonstrated that T-ALL cell lines show differential sensitivity to inhibition at different nodes in the PI3K/AKT/mTOR pathway and inhibiting AKT or mTOR may have a therapeutic benefit in this disease setting. PMID:26989080

  8. Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.

    PubMed

    Coulonval, K; Vandeput, F; Stein, R C; Kozma, S C; Lamy, F; Dumont, J E

    2000-06-01

    The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action. PMID:10816429

  9. [Changes in the phosphoinositide metabolism in the blood and tissues of benign and malignant uterine tumors].

    PubMed

    Damirov, M M; Sliusar', N N; Kulakov, V I; Bakuleva, I P; Matruk, T A

    1995-01-01

    Measurements of phosphoinositide levels in the blood, immunocompetent cells, and tumors of 105 patients with uterine myomas, 24 patients with cancer of the corpus uteri, and 17 ones with uterine sarcoma showed that the parameters of phosphoinositide metabolism in the blood of patients with tumors of the uterus reliably differed from those in healthy women. The content of phosphatidylinosites and other phosphoinositide fractions in patients with uterine myomas reliably differed from those in patients with malignant tumors of the uterus, this permitting the use of such measurements in the differential diagnosis. Phosphoinositide mechanism of development of tumors of the uterus is discussed, which is related to the "new" phosphoinositides and secondary messengers directly participating in transfer of cell growth signals. PMID:7785738

  10. The dual pathway inhibitor rigosertib is effective in direct-patient tumor xenografts of head and neck squamous cell carcinomas

    PubMed Central

    Anderson, Ryan T.; Keysar, Stephen B.; Bowles, Daniel W.; Glogowska, Magdalena J.; Astling, David P.; Morton, J. Jason; Le, Phuong; Umpierrez, Adrian; Eagles-Soukup, Justin; Gan, Gregory N.; Vogler, Brian W.; Sehrt, Daniel; Takimoto, Sarah M.; Aisner, Dara L.; Wilhelm, Francois; Frederick, Barbara A.; Varella-Garcia, Marileila; Tan, Aik-Choon; Jimeno, Antonio

    2013-01-01

    The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in head and neck squamous cell cancer (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from 8 HNSCC patients (4 HPV16-positive). HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, western blotting, and IHC. Rigosertib had potent antiproliferative effects on 11 of the 16 HPV− HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in 3/8 HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA activating event (amplification or mutation) and a p53 inactivating event (either HPV16-mediated or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV+ and HPV− HNSCCs focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient for rigosertib-sensitivity. PMID:23873848

  11. Roles of cell signaling pathways in cell-to-cell contact-mediated Epstein-Barr virus transmission.

    PubMed

    Nanbo, Asuka; Terada, Haruna; Kachi, Kunihiro; Takada, Kenzo; Matsuda, Tadashi

    2012-09-01

    Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells. PMID:22718812

  12. Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

    NASA Technical Reports Server (NTRS)

    Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

    2003-01-01

    In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

  13. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    SciTech Connect

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M.

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  14. Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

    PubMed Central

    Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

  15. Endothelin stimulates phosphoinositide hydrolysis and PAF synthesis in brain microvessels.

    PubMed

    Catalán, R E; Martínez, A M; Aragonés, M D; Martínez, A; Díaz, G

    1996-11-01

    Treatment of brain microvessels with the three endothelin (ET) isoforms resulted in an increase of phosphoinositide turnover by activation of phospholipase C in a dose- and time-dependent manner. Both ET-1 and ET-2 are maximally effective, whereas the effect evoked by ET-3 was smaller. Concomitantly, there was an enhanced production of a platelet-activating factor (PAF)-like material. This was identified by standard and biological probes in platelets, such as induction of aggregation, phosphatidic acid (PA) production, increase of endogenous protein phosphorylation, and reversal of these responses by a PAF antagonist. The effects evoked by endothelins on phosphoinositide metabolism and PAF production were, to a certain extent, dependent on the presence of extracellular Ca2+. In addition, ET induced changes in Ca2+ dynamics, evoking an initial and rapid intracellular mobilization and influx of Ca2+ and, later, a maintained Ca2+ influx. These findings contribute to the understanding of the pathophysiological role of ET in the blood-brain barrier (BBB). PMID:8898708

  16. Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo.

    PubMed

    Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M; Dong, Zigang; Lee, Hyong Joo

    2010-05-01

    Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1alpha expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70(S6K) in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

  17. Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation

    SciTech Connect

    Kim, Ji Eun; Shepherd, Peter R. Chaussade, Claire

    2009-02-20

    PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110{alpha} and p110{delta} and that after differentiation, p110{delta} levels fall while p110{alpha} levels rise, together with C/EBP{alpha} and PPAR{gamma}. When using specific inhibitors during the differentiation process, we observed that neither p110{beta} nor p110{delta} inhibition, had any significant effect. In contrast PIK-75, a selective p110{alpha} inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110{alpha} inhibitors could potentially have a severe impact on fat cell numbers in vivo.

  18. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.

    PubMed

    Maya-Monteiro, Clarissa M; Almeida, Patricia E; D'Avila, Heloisa; Martins, Aline S; Rezende, Ana Paula; Castro-Faria-Neto, Hugo; Bozza, Patricia T

    2008-01-25

    Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases. PMID:18039669

  19. An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.

    PubMed

    Guo, Deliang; Reinitz, Felicia; Youssef, Mary; Hong, Cynthia; Nathanson, David; Akhavan, David; Kuga, Daisuke; Amzajerdi, Ali Nael; Soto, Horacio; Zhu, Shaojun; Babic, Ivan; Tanaka, Kazuhiro; Dang, Julie; Iwanami, Akio; Gini, Beatrice; Dejesus, Jason; Lisiero, Dominique D; Huang, Tiffany T; Prins, Robert M; Wen, Patrick Y; Robins, H Ian; Prados, Michael D; Deangelis, Lisa M; Mellinghoff, Ingo K; Mehta, Minesh P; James, C David; Chakravarti, Arnab; Cloughesy, Timothy F; Tontonoz, Peter; Mischel, Paul S

    2011-10-01

    Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients. PMID:22059152

  20. Dimethyl Cardamonin Exhibits Anti-inflammatory Effects via Interfering with the PI3K-PDK1-PKCα Signaling Pathway

    PubMed Central

    Yu, Wan-Guo; He, Hao; Yao, Jing-Yun; Zhu, Yi-Xiang; Lu, Yan-Hua

    2015-01-01

    Consumption of herbal tea [flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae)] is associated with health beneficial effects against multiple diseases including diabetes, asthma, and inflammatory bowel disease. Emerging evidences have reported that High mobility group box 1 (HMGB1) is considered as a key “late” proinflammatory factor by its unique secretion pattern in aforementioned diseases. Dimethyl cardamonin (2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone, DMC) is a major ingredient of C. operculatus flower buds. In this study, the anti-inflammatory effects of DMC and its underlying molecular mechanisms were investigated on lipopolysaccharide (LPS)-induced macrophages. DMC notably suppressed the mRNA expressions of TNF-α, IL-1β, IL-6, and HMGB1, and also markedly decreased their productions in a time- and dose-dependent manner. Intriguingly, DMC could notably reduce LPS-stimulated HMGB1 secretion and its nucleo-cytoplasmic translocation. Furthermore, DMC dose-dependently inhibited the activation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), and protein kinase C alpha (PKCα). All these data demonstrated that DMC had anti-inflammatory effects through reducing both early (TNF-α, IL-1β, and IL-6) and late (HMGB1) cytokines expressions via interfering with the PI3K-PDK1-PKCα signaling pathway. PMID:26535080

  1. VEGF Silencing Inhibits Human Osteosarcoma Angiogenesis and Promotes Cell Apoptosis via PI3K/AKT Signaling Pathway.

    PubMed

    Zhao, Jian; Zhang, Zi-Ru; Zhao, Na; Ma, Bao-An; Fan, Qing-Yu

    2015-11-01

    Vascular endothelial growth factor (VEGF) is one of the most effective angiogenic factors that promote generation of tumor vasculature. VEGF is usually up-regulated in multiple cancers including osteosarcoma and glioma. To further explore the potential molecular mechanism that inhibits tumor growth induced by interference of VEGF expression, we constructed a Lv-shVEGF vector and assessed the efficiency of VEGF silencing and its influence in U2OS cells. The data demonstrate that Lv-shVEGF has high inhibition efficiency on VEGF expression, which inhibits proliferation and promotes apoptosis of U2OS cells in vitro. Our results also indicate that inhibition of VEGF expression suppresses osteosarcoma tumor growth in vivo and reduces osteosarcoma angiogenesis. We also found that the activations of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) were considerably reduced after osteosarcoma cells were treated with Lv-shVEGF. Taken together, our data demonstrate that VEGF silencing suppresses cell proliferation, promotes cell apoptosis, and reduces osteosarcoma angiogenesis through inactivation of PI3K/AKT signaling pathway. PMID:27352347

  2. A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

    PubMed

    Tang, G; Dong, X; Huang, X; Huang, X-J; Liu, H; Wang, Y; Ye, W-C; Shi, L

    2015-09-10

    Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo. PMID:26183020

  3. Shikonin promotes autophagy in BXPC-3 human pancreatic cancer cells through the PI3K/Akt signaling pathway

    PubMed Central

    SHI, SHUQING; CAO, HAIMEI

    2014-01-01

    The present study aimed to investigate the effect of shikonin on autophagy in BXPC-3 human pancreatic cancer cells and its underlying mechanism. Cell viability was assessed using the Cell Counting Kit-8 assay and the expression of light chain (LC) 3, p62, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-PI3K and p-Akt was analyzed using western blot analysis. Following treatment with 1 μmol/l shikonin for 48 h and 2.5 and 5 μmol/l shikonin for 24 and 48 h, the viability of the BXPC-3 cells was found to be significantly reduced and the protein expression of LC3-II/LC3-I was observed to be increased, while the protein expression of p62, PI3K, Akt, p-PI3K and p-Akt was decreased. These findings suggest that shikonin promotes autophagy in BXPC-3 cells and that the underlying mechanism may be associated with the PI3K/Akt signaling pathway. PMID:25120662

  4. A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

    PubMed Central

    Zhou, K; Takegawa, K; Emr, S D; Firtel, R A

    1995-01-01

    Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5

  5. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes

    PubMed Central

    2016-01-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5–15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  6. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes.

    PubMed

    Weinberg, Mark A

    2016-07-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5-15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  7. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  8. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    PubMed

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling. PMID:24068326

  9. Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C.

    PubMed Central

    Dewaste, V; Pouillon, V; Moreau, C; Shears, S; Takazawa, K; Erneux, C

    2000-01-01

    Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin. PMID:11085927

  10. RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.

    PubMed

    Najjar, Malek; Saleh, Danish; Zelic, Matija; Nogusa, Shoko; Shah, Saumil; Tai, Albert; Finger, Joshua N; Polykratis, Apostolos; Gough, Peter J; Bertin, John; Whalen, Michael J; Pasparakis, Manolis; Balachandran, Siddharth; Kelliher, Michelle; Poltorak, Alexander; Degterev, Alexei

    2016-07-19

    Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies. PMID:27396959

  11. A Role for a CXCR2/Phosphatidylinositol 3-Kinase γ Signaling Axis in Acute and Chronic Vascular Permeability▿ †

    PubMed Central

    Gavard, Julie; Hou, Xu; Qu, Yi; Masedunskas, Andrius; Martin, Daniel; Weigert, Roberto; Li, Xuri; Gutkind, J. Silvio

    2009-01-01

    Most proangiogenic polypeptide growth factors and chemokines enhance vascular permeability, including vascular endothelial growth factor (VEGF), the main target for anti-angiogenic-based therapies, and interleukin-8 (IL-8), a potent proinflammatory mediator. Here, we show that in endothelial cells IL-8 initiates a signaling route that converges with that deployed by VEGF at the level of the small GTPase Rac1 and that both act through the p21-activated kinase to promote the phosphorylation and internalization of VE-cadherin. However, whereas VEGF activates Rac1 through Src-related kinases, IL-8 specifically signals to Rac1 through its cognate G protein-linked receptor, CXCR2, and the stimulation of the phosphatidylinositol 3-kinase γ (PI3Kγ) catalytic isoform, thereby providing a specific molecular targeted intervention in vascular permeability. These results prompted us to investigate the potential role of IL-8 signaling in a mouse model for retinal vascular hyperpermeability. Importantly, we observed that IL-8 is upregulated upon laser-induced retinal damage, which recapitulates enhanced vascularization, leakage, and inflammatory responses. Moreover, blockade of CXCR2 and PI3Kγ was able to limit neovascularization and choroidal edema, as well as macrophage infiltration, therefore contributing to reduce retinal damage. These findings indicate that the CXCR2 and PI3Kγ signaling pathway may represent a suitable target for the development of novel therapeutic strategies for human diseases characterized by vascular leakage. PMID:19255141

  12. Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase

    PubMed Central

    Jung, Sung Keun; Jeong, Chul-Ho

    2016-01-01

    Background: Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D. Methods: We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved. Results: DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression. Conclusions: DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth. PMID:27051646

  13. Wnt5a promotes migration of human osteosarcoma cells by triggering a phosphatidylinositol-3 kinase/Akt signals

    PubMed Central

    2014-01-01

    Wnt5a is classified as a non-transforming Wnt family member and plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in osteosarcoma progression remains poorly defined. In this study, we found that Wnt5a stimulated the migration of human osteosarcoma cells (MG-63), with the maximal effect at 100 ng/ml, via enhancing phosphorylation of phosphatidylinositol-3 kinase (PI3K)/Akt. PI3K and Akt showed visible signs of basal phosphorylation and elevated phosphorylation at 15 min after stimulation with Wnt5a. Pharmaceutical inhibition of PI3K with LY294002 significantly blocked the Wnt5a-induced activation of Akt (p-Ser473) and decreased Wnt5a-induced cell migration. Akt siRNA remarkably inhibited Wnt5a-induced cell migration. Additionally, Wnt5a does not alter the total expression and phosphorylation of β-catenin in MG-63 cells. Taken together, we demonstrated for the first time that Wnt5a promoted osteosarcoma cell migration via the PI3K/Akt signaling pathway. These findings could provide a rationale for designing new therapy targeting osteosarcoma metastasis. PMID:24524196

  14. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

    SciTech Connect

    Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta; Durden, Donald L.

    2014-09-10

    -3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo. - Highlights: • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes. • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo. • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.

  15. Regulation of Neuron Survival through an Intersectin-Phosphoinositide 3′-Kinase C2β-AKT Pathway▿ †

    PubMed Central

    Das, Margaret; Scappini, Erica; Martin, Negin P.; Wong, Katy A.; Dunn, Sara; Chen, Yun-Ju; Miller, Stephanie L. H.; Domin, Jan; O'Bryan, John P.

    2007-01-01

    While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3′-kinase C2β (PI3K-C2β) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2β-AKT survival pathway. ITSN associated with PI3K-C2β on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2β activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2β and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival. PMID:17875942

  16. PREX2 promotes the proliferation, invasion and migration of pancreatic cancer cells by modulating the PI3K signaling pathway

    PubMed Central

    Yang, Jianyi; Gong, Xuejun; Ouyang, Lu; He, Wen; Xiao, Rou; Tan, Li

    2016-01-01

    Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchanger factor 2 (PREX2) is a novel regulator of the small guanosine triphosphatase Rac, and has been observed to be implicated in human cancer by inhibiting the activity of phosphatase and tensin homolog (PTEN), thus upregulating the activity of the phosphoinositide 3-kinase (PI3K) signaling pathway. However, the exact role of PREX2 in pancreatic cancer has not been reported to date. In the present study, the expression levels of PREX2 were observed to be frequently increased in pancreatic cancer specimens compared with those in their matched adjacent normal tissues. In addition, PREX2 expression was also frequently upregulated in several pancreatic cancer cell lines, including AsPC-1, BxPC-3, PANC-1 and CFAPC-1, compared with that in the normal pancreatic epithelial cell line HPC-Y5. Overexpression of PREX2 significantly promoted the proliferation, invasion and migration of pancreatic cancer PANC-1 cells, while small interfering RNA-induced knockdown of PREX2 expression significantly inhibited the proliferation, invasion and migration of these cells. Investigation of the molecular mechanism revealed that the overexpression of PREX2 upregulated the phosphorylation levels of PTEN, indicating that the activity of PTEN was reduced, which further increased the phosphorylation levels of AKT, which indicated that the activity of the PI3K signaling pathway was upregulated. By contrast, knockdown of PREX2 upregulated the activity of PTEN and inhibited the activity of the PI3K signaling pathway. In conclusion, the present study demonstrated that PREX2 regulates the proliferation, invasion and migration of pancreatic cancer cells, probably at least via modulation of the activity of PTEN and the PI3K signaling pathway. PMID:27446408

  17. Magnetic stimulation modulates structural synaptic plasticity and regulates BDNF-TrkB signal pathway in cultured hippocampal neurons.

    PubMed

    Ma, Jun; Zhang, Zhanchi; Su, Yuhong; Kang, Lin; Geng, Dandan; Wang, Yanyong; Luan, Feng; Wang, Mingwei; Cui, Huixian

    2013-01-01

    Repetitive transcranial magnetic stimulation (rTMS) is a neuropsychiatric tool that can be used to investigate the neurobiology of learning and cognitive function. Few studies have examined the effects of low frequency (⩽1Hz) magnetic stimulation (MS) on structural synaptic plasticity of neurons in vitro, thus, the current study examined its effects on hippocampal neuron and synapse morphology, as well as synaptic protein markers and signaling pathways. Similarly, both intensities of low frequency magnetic stimulation (1Hz) activated brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) pathways, including the pathways for mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and for phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). Specifically, low intensity magnetic stimulation (LIMS, 1.14Tesla, 1Hz) promoted more extensive dendritic and axonal arborization, as well as increasing synapses density, thickening PSD (post synaptic density) and upregulation of synaptophysin (SYN), growth associated protein 43 (GAP43) and post synaptic density 95 (PSD95). Conversely, high intensity magnetic stimulation (HIMS, 1.55Tesla, 1Hz) appeared to be detrimental, reducing dendritic and axonal arborization and causing apparent structural damage, including thinning of PSD, less synapses and disordered synaptic structure, as well as upregulation of GAP43 and PSD95, possibly for their ability to mitigate dysfunction. In conclusion, we infers that low frequency magnetic stimulation participates in regulating structural synaptic plasticity of hippocampal neurons via the activation of BDNF-TrkB signaling pathways. PMID:23201339

  18. Analysis of hormone-induced changes of phosphoinositide metabolism in rat liver

    SciTech Connect

    Wallace, M.A.; Fain, J.N.

    1985-01-01

    The relationship between hormone-stimulated phosphoinositide turnover and Ca/sup 2 +/ flux can be investigated using radiolabelled hepatocytes and the subcellular fractions derived from them or from whole liver. Comparison of the results obtained using intact cells to those from subcellular fractions should ultimately lead to a reconstruction of the transmembrane signaling events through which hormone such as vasopressin, angiotensin, and catecholamines acutely activate liver glycogenolysis. The paper reviews hormone-stimulated phosphoinositide metabolism in intact hepatocytes as well as hepatic enzymes involved in phosphoinositide metabolism. Also discussed is the current status of studies on hormone action in broken cell preparations in liver.

  19. Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF

    PubMed Central

    Mercer, Paul F; Woodcock, Hannah V; Eley, Jessica D; Platé, Manuela; Sulikowski, Michal G; Durrenberger, Pascal F; Franklin, Linda; Nanthakumar, Carmel B; Man, Yim; Genovese, Federica; McAnulty, Robin J; Yang, Shuying; Maher, Toby M; Nicholson, Andrew G; Blanchard, Andy D; Marshall, Richard P; Lukey, Pauline T; Chambers, Rachel C

    2016-01-01

    Rationale Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. Methods We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. Results We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. Conclusions Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. Trial registration number NCT01725139, pre-clinical. PMID:27103349

  20. Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

    PubMed

    Eller-Borges, Roberta; Batista, Wagner L; da Costa, Paulo E; Tokikawa, Rita; Curcio, Marli F; Strumillo, Scheilla T; Sartori, Adriano; Moraes, Miriam S; de Oliveira, Graciele A; Taha, Murched O; Fonseca, Fábio V; Stern, Arnold; Monteiro, Hugo P

    2015-05-01

    The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK. PMID:25819133

  1. Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

    PubMed Central

    Devereaux, Kelly; Ogasawara, Yuta; Zhou, Xiang; Wang, Fan; Yamamoto, Akitsugu; De Camilli, Pietro; Di Paolo, Gilbert

    2013-01-01

    Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. PMID:24098492

  2. Deciphering the roles of phosphoinositide lipids in phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Haas, Albert

    2016-01-01

    ABSTRACT Professional phagocytes engulf microbial invaders into plasma membrane-derived phagosomes. These mature into microbicidal phagolysosomes, leading to killing of the ingested microbe. Phagosome maturation involves sequential fusion of the phagosome with early endosomes, late endosomes, and the main degradative compartments in cells, lysosomes. Some bacterial pathogens manipulate the phosphoinositide (PIP) composition of phagosome membranes and are not delivered to phagolysosomes, pointing at a role of PIPs in phagosome maturation. This hypothesis is supported by comprehensive microscopic studies. Recently, cell-free reconstitution of fusion between phagosomes and endo(lyso)somes identified phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 3-phosphate [PI(3)P] as key regulators of phagolysosome biogenesis. Here, we describe the emerging roles of PIPs in phagosome maturation and we present tools to study PIP involvement in phagosome trafficking using intact cells or purified compartments. PMID:27489580

  3. Phosphoinositide kinase signaling controls ER-PM cross-talk

    PubMed Central

    Omnus, Deike J.; Manford, Andrew G.; Bader, Jakob M.; Emr, Scott D.; Stefan, Christopher J.

    2016-01-01

    Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca2+-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions. PMID:26864629

  4. Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils.

    PubMed

    Grandordy, B M; Lacroix, H; Mavoungou, E; Krilis, S; Crea, A E; Spur, B W; Lee, T H

    1990-03-30

    Lipoxins (LX) are trihydroxytetraene metabolites derived from arachidonic acid via an interaction between the 5- and 15-lipoxygenases. Preincubation of [3H] myo-inositol labeled PMN with 10-7M and 10-5M LXA4 for 1 minute at 37 degrees C resulted in a concentration dependent inhibition of the generation of [3H] IP3 and [3H] IP in cells subsequently stimulated by increasing doses of LTB4 or FMLP for 1 minute at 37 degrees C. Preincubation of PMN with LXA4 did not inhibit specific binding of [3H] LTB4 to PMN. These results indicate that LXA4 inhibits chemotactic factor-induced phosphoinositide hydrolysis at a post-receptor level. PMID:2157419

  5. In vivo tracking of phosphoinositides in Drosophila photoreceptors

    PubMed Central

    Hardie, Roger C.; Liu, Che-Hsiung; Randall, Alexander S.; Sengupta, Sukanya

    2015-01-01

    ABSTRACT In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle. PMID:26483384

  6. Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways.

    PubMed

    Wu, Kang-Ni; Zhao, Yan-Min; He, Ying; Wang, Bin-Sheng; Du, Kai-Li; Fu, Shan; Hu, Kai-Min; Zhang, Li-Fei; Liu, Li-Zhen; Hu, Yong-Xian; Wang, Ying-Jia; Huang, He

    2014-03-01

    T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies. PMID:23741975

  7. Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

    PubMed

    Roy, Avik; Modi, Khushbu K; Khasnavis, Saurabh; Ghosh, Supurna; Watson, Richard; Pahan, Kalipada

    2014-01-01

    Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias. PMID:25007337

  8. Enhancement of Morphological Plasticity in Hippocampal Neurons by a Physically Modified Saline via Phosphatidylinositol-3 Kinase

    PubMed Central

    Roy, Avik; Modi, Khushbu K.; Khasnavis, Saurabh; Ghosh, Supurna; Watson, Richard; Pahan, Kalipada

    2014-01-01

    Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer’s disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias. PMID:25007337

  9. Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

    PubMed Central

    Cuesta-Geijo, Miguel A.; Galindo, Inmaculada; Hernáez, Bruno; Quetglas, Jose Ignacio; Dalmau-Mena, Inmaculada; Alonso, Covadonga

    2012-01-01

    Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P2). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection. PMID:23133661

  10. Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation.

    PubMed

    Foukas, Lazaros C; Claret, Marc; Pearce, Wayne; Okkenhaug, Klaus; Meek, Stephen; Peskett, Emma; Sancho, Sara; Smith, Andrew J H; Withers, Dominic J; Vanhaesebroeck, Bart

    2006-05-18

    The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers. PMID:16625210

  11. Capitalizing on tumor genotyping: Towards the design of mutation specific inhibitors of phosphoinsitide-3-kinase

    SciTech Connect

    Gabelli, Sandra B.; Duong-Ly, Krisna C.; Brower, Evan T.; Amzel, L. Mario

    2011-09-06

    PI3Ks catalyze the phosphorylation of the inositol hydroxyls of phosphoinositide membrane components. The changes in phosphorylation of the inositides recruit proteins to the plasma membrane that initiate important signaling cascades. PI3K{alpha}, one of the class IA PI3Ks, is highly mutated in cancers. All mutations analyzed result in an increase in enzymatic activity. The structures of this enzyme determined by X-ray diffraction, provide a framework for analyzing the possible structural effect of these mutations and their effect on the enzymatic activity. Many of the mutations occur at domain interfaces where they can affect domain interactions and relieve the inhibition of the wild-type enzyme by the nSH2 domain of p85. This mechanism is analogous to the mechanism of physiological activation by activated tyrosine-kinase receptors in which the phosphorylated tyrosine of the receptor (or their substrates) dislodges the nSH2 from its inhibitory position in the complex by competing with its binding to a loop in the helical domain. Other mutations in the kinase domain can directly affect the conformation of the catalytic site. One mutation, His1047Arg, uses a completely different mechanism: it changes the conformation of the C-terminal loop in such a way that it increases the interaction of the enzyme with the membrane, granting increased access to the phosphoinositide substrates. Taking advantage of the reliance of some cancers on the increased activity of mutated PI3K{alpha}, will require the development of isoform-specific, mutant-specific inhibitors. The structural, biochemical and physiological data that are becoming available for PI3Ks are an important first step in this direction.

  12. The Phox homology (PX) domain, a new player in phosphoinositide signalling.

    PubMed Central

    Xu, Y; Seet, L F; Hanson, B; Hong, W

    2001-01-01

    Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins. PMID:11736640

  13. Sedanolide induces autophagy through the PI3K, p53 and NF-κB signaling pathways in human liver cancer cells.

    PubMed

    Hsieh, Shu-Ling; Chen, Chi-Tsai; Wang, Jyh-Jye; Kuo, Yu-Hao; Li, Chien-Chun; Hsieh, Lan-Chi; Wu, Chih-Chung

    2015-12-01

    Sedanolide (SN), a phthalide-like compound from celery seed oil, possesses antioxidant effects. However, the effect of SN on cell death in human liver cancer cells has yet to be determined. In this study, cell viability determination, monodansylcadaverine (MDC) fluorescent staining and immunoblot analysis were performed to determine autophagy induction and autophagy-induced protein expression changes via molecular examination after human liver cancer (J5) cells were treated with SN. Our studies demonstrate that SN suppressed J5 cell viability by inducing autophagy. Phosphoinositide 3-kinase (PI3K)-I, mammalian target of rapamycin (mTOR) and Akt protein levels decreased, whereas PI3K-III, LC3-II and Beclin-1 protein levels increased following SN treatment in J5 cells. In addition, SN treatment upregulated nuclear p53 and damage-regulated autophagy modulator (DRAM) and downregulated cytosolic p53 and Tp53-induced glycolysis and apoptosis regulator (TIGAR) expression in J5 cells. Furthermore, the cytosolic phosphorylation of inhibitor of kappa B (IκB) and nuclear p65 and the DNA-binding activity of NF-κB increased after SN treatment. These results suggest that SN induces J5 cell autophagy by regulating PI3K, p53 and NF-κB autophagy-associated signaling pathways in J5 cells. PMID:26500073

  14. Methyl 3,4-dihydroxybenzoate promote rat cortical neurons survival and neurite outgrowth through the adenosine A2a receptor/PI3K/Akt signaling pathway.

    PubMed

    Zhang, Zheng; Cai, Liang; Zhou, Xiaowen; Su, Chaofen; Xiao, Fei; Gao, Qin; Luo, Huanmin

    2015-04-15

    Methyl 3,4-dihydroxybenzoate (MDHB), a kind of phenolic acid compounds, has been reported to have antioxidant effects. Moreover, our previous study found that it could promote neurite outgrowth and brain-derived neurotrophic factor expression in cortical neurons of neonatal rats. In the present study, we focused on the mechanism of its neurotrophic effect; the results showed that MDHB-induced upregulation of neuronal survival and neurite outgrowth in cultured primary cortical neurons could be blocked by the adenosine A2a receptor inhibitor (ZM241385) and the phosphoinositide 3-kinase (PI3K) inhibitor (LY294002). Subsequently, we found that the upregulation of Akt phosphorylation by MDHB could be suppressed by A2a-R and PI3K-specific inhibitor, but not the Trk-R inhibitor. Furthermore, MDHB could activate Akt in a concentration-dependent manner. These results suggested that activation of the PI3K/Akt signaling pathway may be involved in the MDHB-induced neurotrophic effects and MDHB could be a candidate compound to develop drugs for neurodegenerative disease. PMID:25807175

  15. S-Propargyl-cysteine Exerts a Novel Protective Effect on Methionine and Choline Deficient Diet-Induced Fatty Liver via Akt/Nrf2/HO-1 Pathway

    PubMed Central

    Li, Wenwen; Ma, Fenfen; Zhang, Laiyin; Huang, Yong; Li, Xinghui; Zhang, Aijie; Hou, Cuilan; Zhu, Yichun; Zhu, YiZhun

    2016-01-01

    This study investigated the antioxidative effect of S-propargyl-cysteine (SPRC) on nonalcoholic fatty liver (NAFLD) by treating mice fed a methionine and choline deficient (MCD) diet with SPRC for four weeks. We found that SPRC significantly reduced hepatic reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels. Moreover, SPRC also increased the superoxide dismutase (SOD) activity. By Western blot, we found that this protective effect of SPRC was importantly attributed to the regulated hepatic antioxidant-related proteins, including protein kinase B (Akt), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and cystathionine γ-lyase (CSE, an enzyme that synthesizes hydrogen sulfide). Next, we examined the detailed molecular mechanism of the SPRC protective effect using oleic acid- (OA-) induced HepG2 cells. The results showed that SPRC significantly decreased intracellular ROS and MDA levels in OA-induced HepG2 cells by upregulating the phosphorylation of Akt, the expression of HO-1 and CSE, and the translocation of Nrf2. SPRC-induced HO-1 expression and Nrf2 translocation were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Moreover, the antioxidative effect of SPRC was abolished by CSE inhibitor DL-propargylglycine (PAG) and HO-1 siRNA. Therefore, these results proved that SPRC produced an antioxidative effect on NAFLD through the PI3K/Akt/Nrf2/HO-1 signaling pathway. PMID:27313828

  16. Dihydroartemisinin prevents liver fibrosis in bile duct ligated rats by inducing hepatic stellate cell apoptosis through modulating the PI3K/Akt pathway.

    PubMed

    Chen, Qin; Chen, Lianyun; Wu, Xiafei; Zhang, Feng; Jin, Huanhuan; Lu, Chunfeng; Shao, Jiangjuan; Kong, Desong; Wu, Li; Zheng, Shizhong

    2016-03-01

    As a frequent event following chronic insult, liver fibrosis triggers wound healing reactions, with extracellular matrix components accumulated in the liver. During liver fibrogenesis, activation of hepatic stellate cells (HSCs) is the pivotal event. Fibrosis regression can feasibly be treated through pharmacological induction of HSC apoptosis. Herein we showed that dihydroartemisinin (DHA) improved liver histological architecture, decreased hepatic enzyme levels, and inhibited HSCs activation in the fibrotic rat liver. DHA also induced apoptosis of HSCs in such liver, as demonstrated by reduced distribution of α-SMA-positive cells and the presence of high number of cleaved-caspase-3-positive cells in vivo, as well as by down-regulation of Bcl-2 and up-regulation of Bax. In addition, in vitro experiments showed that DHA significantly inhibited HSC proliferation and led to dramatic morphological alterations in HSCs. we found that DHA disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that DHA induced HSC apoptosis through disrupting the phosphoinositide 3-kinase (PI3K)/Akt pathway and that PI3K specific inhibitor LY294002 mimicked the pro-apoptotic effect of DHA. DHA is a promising candidate for the prevention and treatment of liver fibrosis. PMID:26865509

  17. S-Propargyl-cysteine Exerts a Novel Protective Effect on Methionine and Choline Deficient Diet-Induced Fatty Liver via Akt/Nrf2/HO-1 Pathway.

    PubMed

    Li, Wenwen; Ma, Fenfen; Zhang, Laiyin; Huang, Yong; Li, Xinghui; Zhang, Aijie; Hou, Cuilan; Zhu, Yichun; Zhu, YiZhun

    2016-01-01

    This study investigated the antioxidative effect of S-propargyl-cysteine (SPRC) on nonalcoholic fatty liver (NAFLD) by treating mice fed a methionine and choline deficient (MCD) diet with SPRC for four weeks. We found that SPRC significantly reduced hepatic reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels. Moreover, SPRC also increased the superoxide dismutase (SOD) activity. By Western blot, we found that this protective effect of SPRC was importantly attributed to the regulated hepatic antioxidant-related proteins, including protein kinase B (Akt), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and cystathionine γ-lyase (CSE, an enzyme that synthesizes hydrogen sulfide). Next, we examined the detailed molecular mechanism of the SPRC protective effect using oleic acid- (OA-) induced HepG2 cells. The results showed that SPRC significantly decreased intracellular ROS and MDA levels in OA-induced HepG2 cells by upregulating the phosphorylation of Akt, the expression of HO-1 and CSE, and the translocation of Nrf2. SPRC-induced HO-1 expression and Nrf2 translocation were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Moreover, the antioxidative effect of SPRC was abolished by CSE inhibitor DL-propargylglycine (PAG) and HO-1 siRNA. Therefore, these results proved that SPRC produced an antioxidative effect on NAFLD through the PI3K/Akt/Nrf2/HO-1 signaling pathway. PMID:27313828

  18. Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway

    PubMed Central

    Tseng, Chin-Kai; Lin, Chun-Kuang; Hsu, Yao-Chin; Chen, Yen-Hsu; Lee, Jin-Ching

    2016-01-01

    Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC50 value of 5.7 ± 0.2 μM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication. PMID:27023634

  19. Visfatin attenuates the ox-LDL-induced senescence of endothelial progenitor cells by upregulating SIRT1 expression through the PI3K/Akt/ERK pathway.

    PubMed

    Ming, Guang-Feng; Tang, Yong-Jun; Hu, Kai; Chen, Yao; Huang, Wei-Hua; Xiao, Jian

    2016-08-01

    Endothelial progenitor cells (EPCs) play an important role in aging-associated senescence, thereby potentially contributing to vascular pathologies. Visfatin, identified as a new adipocytokine, is closely associated with the senescence of human cells. However, the effects of visfatin on the oxidized low-density lipoprotein (ox-LDL)-induced senescence of EPCs has not yet been explored, to the best of our knowledge. For this purpose, in the present study, we examined the effects of visfatin in ox-LDL-stimulated EPCs as well as the underlying mechanism responsible for these effects. We found that visfatin attenuated the ox-LDL-induced senescence of EPCs by repressing β-galactosidase expression and recovering telomerase activity. Western blot analysis confirmed that visfatin induced a dose-dependent increase in sirtuin 1 (SIRT1) expression in EPCs and ox-LDL exposure decreased SIRT1 expression. Silencing SIRT1 abolished the inhibition of EPC senescence and the suppression of p53 expression induced by visfatin. Moreover, visfatin attenuated the inhibition of phosphorylation of Akt, phosphoinositide-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) induced by ox-LDL. Taken together, these findings suggest that the treatment of EPCs with visfatin markedly attenuates the ox-LDL-induced senescence of EPCs by upregulating SIRT1 expression through the PI3K/Akt/ERK pathway. PMID:27277186

  20. ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

    PubMed Central

    Wang, D.; Guo, T.Q.; Wang, Z.Y.; Lu, J.H.; Liu, D.P.; Meng, Q.F.; Xie, J.; Zhang, X.L.; Liu, Y.; Teng, L.S.

    2014-01-01

    The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases. PMID:25075574

  1. Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway.

    PubMed

    Yu, Jung-Sheng; Chen, Wei-Chun; Tseng, Chin-Kai; Lin, Chun-Kuang; Hsu, Yao-Chin; Chen, Yen-Hsu; Lee, Jin-Ching

    2016-01-01

    Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC50 value of 5.7 ± 0.2 μM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication. PMID:27023634

  2. Sanguinarine Induces Apoptosis of Human Oral Squamous Cell Carcinoma KB Cells via Inactivation of the PI3K/Akt Signaling Pathway.

    PubMed

    Lee, Tae Kyung; Park, Cheol; Jeong, Soon-Jeong; Jeong, Moon-Jin; Kim, Gi-Young; Kim, Wun-Jae; Choi, Yung Hyun

    2016-08-01

    Preclinical Research Sanguinarine, an alkaloid isolated from the root of Sanguinaria canadensis and other plants of the Papaveraceae family, selectively induces apoptotic cell death in a variety of human cancer cells, but its mechanism of action requires further elaboration. The present study investigated the pro-apoptotic effects of sanguinarine in human oral squamous cell carcinoma KB cells. Sanguinarine treatment increased DR5/TRAILR2 (death receptor 5/TRAIL receptor 2) expression and enhanced the activation of caspase-8 and cleavage of its substrate, Bid. Sanguinarine also induced the mitochondrial translocation of pro-apopt